Recombinant gelatins in vaccines

ABSTRACT

The present invention relates to vaccines comprising recombinant gelatin, to methods of producing and using such vaccines, and to vaccination kits.

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/710,249, filed 10 Nov. 2000 which claims the benefit of U.S.Provisional Patent Application Nos. 60/204,437, filed 15 May 2000, and60/165,114, filed 12 Nov. 1999, the specifications of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] This invention relates to vaccines, and, specifically, to vaccineformulations comprising recombinant gelatin.

BACKGROUND OF THE INVENTION

[0003] Vaccines are preparations of killed or modified microorganisms,living attenuated organisms, or living fully virulent organisms, or anyother infective agent, including, but not limited to peptides, proteins,biological macromolecules, or nucleic acids, natural, synthetic, orsemi-synthetic, capable of stimulating an immune response whenadministered to a subject. Vaccines are provided in order to prevent ortemper the severity of subsequent exposure to or infection with asimilar microorganism. In live attenuated vaccines, the microorganismhas typically been treated to produce an avirulent strain capable ofinducing protective immunity. In inactivated vaccines, the infectiousmicrobial nucleic acid components are destroyed prior to administration,without affecting the antigenicity or immunogenecity of the viral coator bacterial outer membrane proteins of the microorganism.

[0004] Vaccination has accomplished radical changes in global health.For example, in 1980, the World Health Organization declared smallpoxeradicated as a result of international vaccination efforts. Diphtheria,whooping cough, and measles, responsible for high rates of childhoodmortality, are the focus of established infant and childhoodimmunization programs in industrialized countries, and there arecontinuing efforts to establish such programs in developing countries.Immunizations protect children against diseases including polio,measles, mumps, rubella (German measles), diphtheria, pertussis(whooping cough), chicken pox, hepatitis B, and Haemophilus influenzaetype b. Current immunization schedules can start within 12 hours ofbirth, and require multiple immunizations during the first two years.Administration of vaccines can occur at various points throughout aperson's lifetime, for example, seasonal vaccines, e.g., “flu vaccine,”and vaccines administered to travelers, etc.

[0005] Vaccines can be administered in a number of ways. Currently,vaccines are primarily delivered through injection or oraladministration. Vaccine formulations can include, for example, adjuvantsto enhance the immune response, so that less vaccine is needed toproduce the desired protective immune response. Various excipients andcarriers, contributing to the consistency and deliverability of thevaccine, can also be provided.

[0006] Vaccines can include various components included within thevaccine formulation to maintain the stability of the vaccine. Suchstabilizers are intended to maintain the integrity of the vaccine,preserving the vaccine's ability to elicit a protective immune responseupon administration. Stabilizers used in vaccine formulations include,but are not limited to, chemical and biological agents that perform avariety of functions, such as, for example, detergents, buffers, salts,sugars, and various protein components, including gelatin, specifically,hydrolyzed gelatin.

Manufacture of Gelatin

[0007] Gelatin is a derivative of collagen, a principal structural andconnective protein in animals. Gelatin is derived from denaturation ofcollagen and contains polypeptide sequences having Gly-X-Y repeats,where X and Y are most often proline and hydroxyproline residues. Thesesequences contribute to triple helical structure and affect the gellingability of gelatin polypeptides. Currently available gelatin isextracted through processing of animal hides and bones, typically frombovine and porcine sources. The biophysical properties of gelatin makeit a versatile material, widely used in a variety of applications andindustries. Gelatin is used, for example, in numerous pharmaceutical andmedical, photographic, industrial, cosmetic, and food and beverageproducts and processes of manufacture. Gelatin is thus a commerciallyvaluable and versatile product.

[0008] Gelatin is typically manufactured from naturally occurringcollagen in bovine and porcine sources, in particular, from hides andbones. In some instances, gelatin can be extracted from, for example,piscine, chicken, or equine sources. Raw materials of typical gelatinproduction, such as bovine hides and bones, originate from animalssubject to government-certified inspection and passed fit for humanconsumption. There is concern over the infectivity of this raw material,due to the presence of contaminating agents such as transmissiblespongiform encephalopathies (TSEs), particularly bovine spongiformencephalopathy (BSE), and scrapie, etc. (See, e.g., Rohwer, R. G.(1996), Dev Biol Stand 88:247-256.) Such issues are especially criticalto gelatin used in pharmaceutical and medical applications.

[0009] Recently, concern about the safety of these materials, asignificant portion of which are derived from bovine sources, hasincreased, causing various gelatin-containing products to become thefocus of several regulatory measures to reduce the potential risk oftransmission of bovine spongiform encephalopathy (BSE), linked to newvariant Creutzfeldt-Jakob disease (nvCJD), a fatal neurological diseasein humans. There is concern that purification steps currently used inthe processing of extracting gelatin from animal tissues and bones maynot be sufficient to remove the likelihood of infectivity due tocontaminating SE-carrying tissue (i.e., brain tissue, etc.). U.S. andEuropean manufacturers specify that raw material for gelatin to beincluded in animal or human food products or in pharmaceutical, medical,or cosmetic applications must not be obtained from a growing number ofBSE countries. In addition, regulations specify that certain materialse.g., bovine brain tissue, are not used in the production of gelatin.

[0010] Current production processes involve several purification andcleansing steps, and can require harsh and lengthy modes of extraction.The animal hides and bones are treated in a rendering process, and theextracted material is subjected to various chemical treatments,including prolonged exposure to highly acidic or alkaline solutions.Numerous purification steps can involve washing and filtration andvarious heat treatments. Acid demineralization and lime treatments areused to remove impurities such as non-collagenous proteins. Bones mustbe degreased. Additional washing and filtration steps, ion exchanges,and other chemical and sterilizing treatments are added to the processto further purify the material. Furthermore, contaminants and impuritiescan still remain after processing, and the resultant gelatin productmust thus typically be clarified, purified, and often furtherconcentrated before being ready for use.

[0011] Commercial gelatin is generally classified as type A or type B.These classifications reflect the pre-treatment extraction sourcesreceive as part of the extraction process. Type A is generally derivedfrom acid-processed materials, usually porcine hides, and type B isgenerally derived from alkaline- or lime-processed materials, usuallybovine bones (ossein) and hides.

[0012] In extracting type A gelatin, the process generally involvessubjecting fresh or frozen porcine hides to successive washings withwater and treatments with dilute acids. The acid-treated skins arewashed again and are then subject to repeated extraction steps in whichthey are treated with hot water, partially hydrolyzing the collagenpresent. The resultant extracts, dilute solutions of gelatin, arefiltered and evaporated, and the resultant concentrates are allowed tocool or chilled to a gel. The gel is subsequently treated in dryingtunnels, or by continuous dryers or other drying devices.

[0013] In the limed process, type B gelatin is derived from donor hidesand skin trimmings washed and then treated with lime. The lime treatmentcan take as long as from one to three months, and is usually aroundsixty days. The limed hides are washed and treated with dilute acids.The hides are then hydrolyzed with hot water and the resulting extractsare processed as described above for the acid-treatment process.

[0014] Type B gelatin can also be processed from ossein sources. Thehard bones are washed, degreased, and leached with successive treatmentsof dilute acids, such as hydrochloric acid. The acid treatment reactswith the mineral contents of bone, which are removed along with theacidic solution, leaving ossein, or demineralized bones. This organicbone matter, washed free of residual acid, is dried for storage orimmediately limed. After liming, ossein is subsequently treated asdescribed above for the production of gelatin from bovine hides. In allcases, after final filtering, demineralization, concentration, anddrying steps, the resultant gelatin product is divided into batches,subjected to various physical, chemical, and bacteriological tests todetermine grade and purity, and ground and blended according tocommercial requirements. In both type A and B extraction processes, theresultant gelatin product typically comprises a mixture of gelatinmolecules, in sizes of from a few thousand up to several hundredthousand Daltons.

[0015] Fish gelatin, classified as gelling or non-gelling types, andtypically processed as Type A gelatin, is also used in certaincommercial applications. Gelling types are usually derived from theskins of warm water fish, while non-gelling types are typically derivedfrom cold water fish. Fish gelatins have widely varying amino acidcompositions, and differ from animal gelatins in having typically lowerproportions of proline and hydroxyproline residues. In contrast toanimal gelatins, fish gelatins typically remain liquid at much lowertemperatures, even at comparable average molecular weights. As withother animal gelatins, fish gelatin is extracted by treatment andsubsequent hydrolyzation of fish skin. Again, as with animal extractionprocesses, the process of extracting fish gelatin results in a productthat lacks homogeneity.

Gelatin in Vaccines

[0016] Anaphylactic reactions to measles, mumps, and rubella vaccines,and to the combined measles-mumps-rubella (MMR) vaccine, have beenreported. (Sakaguchi and Inouye, 2000, Vaccine, 18:2055-2058; Sakaguchiet al., 1999, J Allergy Clin Immunol, 104:695-699.) Despite speculationthat these allergic reactions were caused by allergy to egg proteinspresent in the vaccines, anaphylactic reactions also have been reportedto occur after administration of the MMR vaccine in children whotolerated eggs. It has been found that most of the reactions to livevaccines are caused by an acquired sensitivity to the bovine gelatinincluded in these vaccines. (See, e.g., Nakayama et al. (1999) J AllergyClin Immunol 103:321-325, and references therein; and Sakaguchi et al.(1999) Immunology, 96:286-290.)

[0017] Studies have revealed that anaphylaxis in children in response tolive attenuated viral vaccines containing gelatin is caused by thegelatin, and that gelatin-containing diphtheria-tetanus-acellularpertussis (DTaP) vaccines appear to sensitize children to gelatin.Specifically, a strong causal relationship has been identified betweenDTaP vaccines containing gelatin, anti-gelatin IgE production, and therisk of anaphylaxis following subsequent immunization with live viralvaccines containing bovine gelatin. (Sakaguchi and Inouye, supra;Nakayama et al. supra.)

[0018] Anaphylactic reactions to MMR vaccines which include gelatin as astabilizer have been reported and have been shown to be caused by thebovine gelatin included in these vaccines. Specifically, IgE reactivityto α1 and α2 chains of bovine type I collagen has been identified inchildren with bovine gelatin allergy. (Sakaguchi et al., supra, andreferences therein). Numerous anaphylactic reactions and some urticarialreactions to gelatin-containing measles, mumps, and rubella vaccineshave been associated with IgE-mediated allergenic responses to gelatin.(Nakayama et al., supra) Specific IgG antibodies to gelatin have beenidentified in children with systemic immediate-type andnonimmediate-type reactions to MMR vaccines, suggesting that the immuneresponse to non-human gelatin plays a role in the pathogenesis ofsystemic reactions to live virus vaccines. (Miyazawa et al. (1999)Vaccine 17:2176-2180; and Kelso (1999) J Aller. Clin Immunol.103:200-202, and references therein.)

[0019] Such gelatin-induced vaccine-specific reactions are all the morecritical in the context of increasing concern relating to the use ofanimal-derived, e.g., bovine-derived, materials intended for human andanimal use, for example, in pharmaceutical applications, andconsumption. Such concerns relating to the safety of bovine-derivedmaterials are directed to the risk of exposure to infectious agents thatmight survive or be introduced in the process of extraction andpurification of gelatin from animal sources. (See, e.g., Asher (1999)Dev. Biol. Stand. 99:41-44; and Verdrager (1999) Lancet 354:1304-1305.)A certain oral polio vaccine used only in the United Kingdom and theRepublic of Ireland was recently withdrawn from use after it wasdetermined that fetal bovine calf serum from the United Kingdom was usedin the manufacture of the vaccine. (BBC News Report, “Polio vaccine inBSE scare,” 20 Oct. 2000; World Health Organization, “WHO PositionStamement on Recall of Evans/Medeva Polio Vaccine in UK,” 20 Oct. 2000.)Concerns over the presence of infective agents, such as TSEs, as well asbacterial and other pathogens and endotoxins which might exist afterextraction, have established a need for safe, non-immunogenic materialthat can be used in place of the materials currently derived from animalsources.

Summary

[0020] Current methods of extraction result in a gelatin product that isa heterogeneous mixture of proteins, containing polypeptides withmolecular weight distributions of varying ranges. It is sometimesnecessary to blend various lots of product in order to obtain a gelatinmixture with the physical properties appropriate for use in a desiredapplication. In addition, it is virtually impossible, using currentextraction methods, to obtain a gelatin free of non-gelatin impurities,e.g., protein, lipid, polysaccharides, etc.

[0021] A more homogeneous product, and one produced by more reproduciblemeans, would be desirable. The availability of a homogeneous materialwith reproducible physical characteristics would be desirable, forexample, in various products and processes, where the availability ofgelatin with specific characteristics, such as a fixed range ofmolecular weight, would allow for a reproducible and controlledperformance. There is thus a need for a reliable and reproducible meansof gelatin production that provides a consistent product with controlledcharacteristics.

[0022] In addition, there are concerns relating to the immunogenicityand infectivity of gelatin-containing products resulting from theanimal-source products and methods of their preparation. (See, e.g.,Sakaguchi and Inouye, supra; Sakaguchi et al., supra; Nakayama et al.,supra; Asher, supra; and Verdrager, supra.) There is thus a need for asource of gelatin other than that currently extracted from bovine,porcine, and other animal sources.

[0023] Gelatin producers and end-users have searched for and tested anumber of natural and synthetic substitutes for the animal-sourcegelatin currently available. Alternatives have been identified for a fewapplications, such as the use of cellulosic raw materials in VCAPScapsules (CAPSUGEL; Morris Plains, N.J.), or the proposed use of non-natural gelatin-like proteins from mouse and rat collagen sequences inphotographic emulsions. (See, e.g., Werten, M. W. et al. (1999) Yeast15:1087-1096; and De Wolf, Anton et al., European Application No.EP1014176A2.) However, for most gelatin-based processes and products,the performance characteristics of this key material have not beenduplicated and substitutes have not been adopted. Thus, there is a needfor a means of producing gelatin in a synthetic and reproducible mannerwherein the resultant product can serve as a rational substitute withthe desired performance characteristics.

[0024] There is a need for a versatile gelatin product from a non-animalsource that is readily adaptable for different uses and that answersexisting health and special concerns. In particular, with respect tovaccines, there is a need for a material that safely minimizes the risksof immunogenecity, antigenicity, and/or infectivity from theanimal-derived products, while serving as an effective stabilizer andcomponent of vaccine formulations.

[0025] The present invention answers these needs by providing auniversal replacement material, obtained recombinantly, appropriate foruse in the extraordinarily diverse spectrum of applications in whichgelatin is currently used. In particular, the present invention providesrecombinant gelatin suitable for use in vaccine formulations. Thepresent materials can be designed to possess properties andcharacteristics desired for specific applications, and can thussubstitute for currently available animal-source gelatins as well asprovide new properties and uses previously unavailable.

SUMMARY OF THE INVENTION

[0026] The present invention is directed to recombinant gelatins and,specifically, to the use of recombinant gelatins in vaccines. Therefore,in one embodiment, the present invention provides a vaccine formulationcomprising recombinant gelatin. In a preferred embodiment, therecombinant gelatin is human gelatin. A further aspect of the presentinvention provides that the recombinant gelatin is non-immunogenic.

[0027] In one embodiment, the present invention provides a vaccineformulation comprising recombinant gelatin, wherein the recombinantgelatin confers stability at ambient temperatures. In anotherembodiment, the gelatin is derived from non-native collagen sequence.

[0028] Formulations comprising specific recombinant gelatins arecontemplated. In one aspect, the recombinant gelatin has a molecularweight range selected from the group consisting of about 0 to 50 kDa,about 10 to 30 kDa, about 30 to 50 kDa, about 10 to 70 kDa, about 50 to70 kDa, about 50 to 100 kDa, about 100 to 150 kDa, about 150 to 200 kDa,about 200 to 250 kDa, about 250 to 300 kDa, and about 300 to 350 kDa. Incertain embodiments, the recombinant gelatin has a molecular weightselected from the group consisting of about 1 kDa, about 5 kDa, about 8kDa, about 9 kDa, about 14 kDa, about 16 kDa, about 22 kDa, about 23kDa, about 44 kDa, and about 65 kDa.

[0029] The present invention provides, in one embodiment, a vaccineformulation comprising recombinant gelatin derived from one collagenfree of any other type of collagen. In various aspects, a vaccineformulation comprising recombinant gelatin is provided, wherein therecombinant gelatin is produced by processing of recombinant collagen,or is produced directly from an altered collagen construct.

[0030] In specific embodiments, the present invention encompassesvaccine formulations comprising a sequence sequence selected from thegroup consisting of SEQ ID NOs:15 through 25, 30, 31, and 33.

[0031] The vaccine formulations of the present invention can be suitablefor various modes of delivery, including delivery by injection, nasaldelivery, oral delivery, transdermal delivery, and deep lung delivery.The vaccine formulations of the present invention can be formulated in anumber of ways, for example, in liquid, dry, powdered, spray, andinhalant form. In various embodiments, the present vaccine formulationscan comprise live, inactivated, subunit, single dosage, multiple dosage,conjugate, nucleic acid, DNA, combined, and acellular vaccines. Specificvaccines are contemplated, including, but not limited to, vaccinesformulated for the prevention of a disease selected from the groupconsisting of vacinnia virus (small pox), polio virus (Salk and Sabin),mumps, measles, rubella, diphtheria, tetanus, Varicella-Zoster (chickenpox/shingles), pertussis (whopping cough), Bacille Calmette-Guerin (BCG,tuberculosis), haemophilus influenzae meningitis, rabies, cholera,Japanese encephalitis virus, salmonella typhi, shigella, hepatitis A,hepatitis B, adenovirus, yellow fever, foot-and-mouth disease, herpessimplex virus, respiratory syncytial virus, rotavirus, Dengue, West Nilevirus, Turkey herpes virus (Marek's Disease), influenza, and anthrax.

[0032] In some embodiments, the present invention provides vaccineformulations comprising recombinant gelatin, wherein the recombinantgelatin has endotoxin levels below 1.000 EU/mg, 0.500 EU/mg, 0.050EU/mg, and 0.005 EU/mg. In one aspect, the present invention encompassesa vaccine formulation comprising recombinant gelatin wherein therecombinant gelatin is proteolytically stable. A vaccine stabilizercomprising recombinant gelatin is specifically contemplated. The presentinvention provides methods for producing vaccine formulations comprisingrecombinant gelatin. In one embodiment, the method comprises providingrecombinant gelatin and providing a vaccine, and combining therecombinant gelatin and the vaccine. A method of inducing an immuneresponse in a subject is also provided, the method comprisingadministering a vaccine comprising recombinant gelatin to the subject.Further, kits comprising a vaccine comprising recombinant gelatin and adelivery device for the vaccine are also contemplated. In variousembodiments, the delivery device is a device suitable, for example, forinjectable, nasal, mucosal, and aerosol delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 sets forth results showing the expression of recombinantgelatins.

[0034]FIGS. 2A and 2B set forth results demonstrating that recombinantgelatins support cell attachment.

[0035]FIG. 3 sets forth results demonstrating the production ofproteolytically stable recombinant gelatins.

[0036]FIG. 4 sets forth results demonstrating the production ofhydroxylated recombinant gelatins.

[0037]FIG. 5 sets forth results showing the purification of recombinantgelatin following in vitro hydroxylation.

[0038]FIG. 6 sets forth results showing the stability of recombinantgelatins expressed in the presence or absence of prolyl 4-hydroxylase.

[0039]FIG. 7 sets forth results demonstrating enhanced recombinantgelatin expression by supplementation of expression media

[0040]FIG. 8 sets forth results comparing commercially availablegelatins to cross-linked recombinant gelatin.

[0041]FIG. 9 sets forth results comparing the molecular weightdistribution of commercially available gelatins.

[0042]FIGS. 10A, 10B, 10C, 10D, 10E, and 10F set forth results showingthe hydrolysis of commercially available gelatins performed at 120° C.

[0043]FIGS. 11A, 11B, 11C, and 11D set forth results showing thehydrolysis of commercially available gelatins performed at 150° C.

[0044]FIGS. 12A and 12B set forth results showing the acid and thermalhydrolysis of recombinant human collagen type I and type III.

[0045]FIG. 13 sets forth results showing the enzymatic hydrolysis ofrecombinant human collagen type I.

[0046]FIG. 14 sets forth a Western blot analysis of recombinant humancollagens and recombinant human gelatins using antisera from Guinea pigsimmunized with recombinant human collagen type I.

[0047]FIGS. 15A and 15B set forth results showing antisera from Guineapigs immunized with recombinant human collagen type I is reactive tospecific cyanogen bromide fragments of collagen type I.

[0048]FIG. 16 sets forth ELISA results showing antisera from Guinea pigsimmunized with recombinant human collagen type I is not reactive torecombinant human gelatins.

DESCRIPTION OF THE INVENTION

[0049] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

[0050] It must be noted that as used herein, and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” is reference to one or more of such host cells andequivalents thereof known to those skilled in the art, and reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0051] Unless defined otherwise, all technical and scientific terms usedherein have the meanings as commonly understood by one of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologies,etc., which are reported in the publications which might be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. Each reference cited herein isincorporated herein by reference in its entirety.

[0052] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of chemistry, biochemistry,molecular biology, immunology and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18^(th)ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods InEnzymology, Academic Press, Inc.; Handbook of Experimental Immunology,Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, BlackwellScientific Publications); Maniatis, T. et al., eds. (1989) MolecularCloning: A Laboratory Manual, 2^(nd) edition, Vols. I-III, Cold SpringHarbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4^(th) edition, John Wiley & Sons; Reamet al., eds. (1998) Molecular Biology Techniques: An IntensiveLaboratory Course, Academic Press); PCR (Introduction to BiotechniquesSeries), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

Definitions

[0053] The term “collagen” refers to any one of the known collagentypes, including collagen types I through XX, as well as to any othercollagens, whether natural, synthetic, semi-synthetic, or recombinant.The term also encompasses procollagens. The term collagen encompassesany single-chain polypeptide encoded by a single polynucleotide, as wellas homotrimeric and heterotrimeric assemblies of collagen chains. Theterm “collagen” specifically encompasses variants and fragments thereof,and functional equivalents and derivatives thereof, which preferablyretain at least one structural or functional characteristic of collagen,for example, a (Gly-X-Y)_(n) domain.

[0054] The term “procollagen” refers to a procollagen corresponding toany one of the collagen types I through XX, as well as to a procollagencorresponding to any other collagens, whether natural, synthetic,semi-synthetic, or recombinant, that possesses additional C-terminaland/or N-terminal propeptides or telopeptides that assist in trimerassembly, solubility, purification, or any other function, and that thenare subsequently cleaved by N-proteinase, C-proteinase, or otherenzymes, e.g., proteolytic enzymes, associated with collagen production.The term procollagen specifically encompasses variants and fragmentsthereof, and functional equivalents and derivatives thereof, whichpreferably retain at least one structural or functional characteristicof collagen, for example, a (Gly-X-Y)n domain.

[0055] “Gelatin” as used herein refers to any gelatin, whether extractedby traditional methods or recombinant or biosynthetic in origin, or toany molecule having at least one structural and/or functionalcharacteristic of gelatin. Gelatin is currently obtained by extractionfrom collagen derived from animal (e.g., bovine, porcine, rodent,chicken, equine, piscine, etc.) sources, for example, bones and tissues.The term gelatin encompasses both the composition of more than onepolypeptide included in a gelatin product, as well as an individualpolypeptide contributing to the gelatin material. Thus, the termrecombinant gelatin as used in reference to the present inventionencompasses both a recombinant gelatin material comprising the presentgelatin polypeptides, as well as an individual gelatin polypeptide ofthe present invention.

[0056] Polypeptides from which gelatin can be derived are polypeptidessuch as collagens, procollagens, and other polypeptides having at leastone structural and/or functional characteristic of collagen. Such apolypeptide could include a single collagen chain, or a collagenhomotrimer or heterotrimer, or any fragments, derivatives, oligomers,polymers, or subunits thereof, containing at least one collagenousdomain (a Gly-X-Y region). The term specifically contemplates engineeredsequences not found in nature, such as altered collagen constructs, etc.An altered collagen construct is a polynucleotide comprising a sequencethat is altered, through deletions, additions, substitutions, or otherchanges, from the naturally occurring collagen gene.

[0057] An “adjuvant” is any agent added to a drug or vaccine toincrease, improve, or otherwise aid its effect. An adjuvant used in avaccine formulation might be an immunological agent that improves theimmune response by producing a non-specific stimulator of the immuneresponse. Adjuvants are often used in non-living vaccines.

[0058] The terms “allele” or “allelic sequence” refer to alternativeforms of genetic sequences. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0059] “Altered” polynucleotide sequences include those with deletions,insertions, or substitutions of different nucleotides resulting in apolynucleotide that encodes the same or a functionally equivalentpolypeptide. Included within this definition are sequences displayingpolymorphisms that may or may not be readily detectable using particularoligonucleotide probes or through deletion of improper or unexpectedhybridization to alleles, with a locus other than the normal chromosomallocus for the subject polynucleotide sequence.

[0060] “Altered” polypeptides may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent polypeptide. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological orimmunological activity of the encoded polypeptide is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0061] “Amino acid” or “polypeptide” sequences or “polypeptides,” asthese terms are used herein, refer to oligopeptide, peptide,polypeptide, or protein sequences, and fragments thereof, and tonaturally occurring or synthetic molecules. Polypeptide or amino acidfragments are any portion of a polypeptide which retains at least onestructural and/or functional characteristic of the polypeptide. In atleast one embodiment of the present invention, polypeptide fragments arethose retaining at least one (Gly-X-Y)_(n) region.

[0062] The term “animal” as it is used in reference, for example, to“animal collagens,” encompasses any collagens, derived from animalsources, whether natural, synthetic, semi-synthetic, or recombinant.Animal sources include, for example, mammalian sources, including, butnot limited to, bovine, porcine, rodent, equine, and ovine sources, andother animal sources, including, but not limited to, chicken and piscinesources, and non-vertebrate sources.

[0063] “Antigenicity” relates to the ability of a substance to, whenintroduced into the body, stimulate the immune response and theproduction of an antibody. An agent displaying the property ofantigenicity is referred to as being antigenic. Antigenic agents caninclude, but are not limited to, a variety of macromolecules such as,for example, proteins, lipoproteins, polysaccharides, nucleic acids,bacteria and bacterial components, and viruses and viral components.

[0064] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides by base-pairing. Forexample, the sequence “A-G-T” binds to the complementary sequence“T-C-A.” Complementarity between two single-stranded molecules may be“partial,” when only some of the nucleic acids bind, or may be complete,when total complementarity exists between the single stranded molecules.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, which depend upon binding between nucleic acidsstrands, and in the design and use, for example, of peptide nucleic acid(PNA) molecules.

[0065] A “deletion” is a change in an amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

[0066] The term “derivative,” as applied to polynucleotides, refers tothe chemical modification of a polynucleotide encoding a particularpolypeptide or complementary to a polynucleotide encoding a particularpolypeptide. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. As used herein to refer topolypeptides, the term “derivative” refers to a polypeptide which ismodified, for example, by hydroxylation, glycosylation, pegylation, orby any similar process. The term “derivatives” encompasses thosemolecules containing at least one structural and/or functionalcharacteristic of the molecule from which it is derived.

[0067] A molecule is said to be a “chemical derivative” of anothermolecule when it contains additional chemical moieties not normally apart of the molecule. Such moieties can improve the molecule'ssolubility, absorption, biological half-life, and the like. The moietiescan alternatively decrease the toxicity of the molecule, eliminate orattenuate any undesirable side effect of the molecule, and the like.Moieties capable of mediating such effects are generally available inthe art and can be found for example, in Remington's PharmaceuticalSciences, supra. Procedures for coupling such moieties to a molecule arewell known in the art.

[0068] An “excipient” as the term is used herein is any inert substanceused as a diluent or vehicle in the formulation of a drug, a vaccine, orother pharmaceutical composition, in order to confer a suitableconsistency or form to the drug, vaccine, or pharmaceutical composition.

[0069] The term “functional equivalent” as it is used herein refers to apolypeptide or polynucleotide that possesses at least one functionaland/or structural characteristic of a particular polypeptide orpolynucleotide. A functional equivalent may contain modifications thatenable the performance of a specific function. The term “functionalequivalent” is intended to include fragments, mutants, hybrids,variants, analogs, or chemical derivatives of a molecule.

[0070] A “fusion protein” is a protein in which peptide sequences fromdifferent proteins are operably linked.

[0071] The term “hybridization” refers to the process by which a nucleicacid sequence binds to a complementary sequence through base pairing.Hybridization conditions can be defined by, for example, theconcentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. Hybridization can occur under conditions ofvarious stringency.

[0072] In particular, stringency can be increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature. For example, for purposes of thepresent invention, hybridization under high stringency conditions occursin about 50% formamide at about 37° C. to 42° C., and under reducedstringency conditions in about 35% to 25% formamide at about 30° C. to35° C. In particular, hybridization occurs in conditions of higheststringency at 42° C. in 50% formamide, 5X SSPE, 0.3% SDS, and 200 μg/mlsheared and denatured salmon sperm DNA.

[0073] The temperature range corresponding to a particular level ofstringency can be further narrowed by methods known in the art, forexample, by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. To removenonspecific signals, blots can be sequentially washed, for example, atroom temperature under increasingly stringent conditions of up to 0.1XSSC and 0.5% SDS. Variations on the above ranges and conditions are wellknown in the art.

[0074] “Immunogenicity” relates to the ability to evoke an immuneresponse within an organism. An agent displaying the property ofimmunogenicity is referred to as being immunogenic. Agents can include,but are not limited to, a variety of macromolecules such as, forexample, proteins, lipoproteins, polysaccharides, nucleic acids,bacteria and bacterial components, and viruses and viral components.Immunogenic agents often have a fairly high molecular weight (usuallygreater than 10 kDa).

[0075] “Infectivity” refers to the ability to be infective or theability to produce infection, referring to the invasion andmultiplication of microorganisms, such as bacteria or viruses within thebody.

[0076] The terms “insertion” or “addition” refer to a change in apolypeptide or polynucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0077] The term “isolated” as used herein refers to a molecule separatednot only from proteins, etc., that are present in the natural source ofthe protein, but also from other components in general, and preferablyrefers to a molecule found in the presence of, if anything, only asolvent, buffer, ion, or other component normally present in a solutionof the same. As used herein, the terms “isolated” and “purified” do notencompass molecules present in their natural source.

[0078] The term “microarray” refers to any arrangement of nucleic acids,amino acids, antibodies, etc., on a substrate. The substrate can be anysuitable support, e.g., beads, glass, paper, nitrocellulose, nylon, orany appropriate membrane, etc. A substrate can be any rigid orsemi-rigid support including, but not limited to, membranes, filters,wafers, chips, slides, fibers, beads, including magnetic or nonmagneticbeads, gels, tubing, plates, polymers, microparticles, capillaries, etc.The substrate can provide a surface for coating and/or can have avariety of surface forms, such as wells, pins, trenches, channels, andpores, to which the nucleic acids, amino acids, etc., may be bound.

[0079] The term “microorganism” can include, but is not limited to,viruses, bacteria, Chlamydia, rickettsias, mycoplasmas, ureaplasmas,fungi, and parasites, including infectious parasites such as protozoans.

[0080] The terms “nucleic acid” or “polynucleotide” sequences or“polynucleotides” refer to oligonucleotides, nucleotides, orpolynucleotides, or any fragments thereof, and to DNA or RNA of naturalor synthetic origin which may be single- or double-stranded and mayrepresent the sense or antisense strand, to peptide nucleic acid (PNA),or to any DNA-like or RNA-like material, natural or synthetic in origin.Polynucleotide fragments are any portion of a polynucleotide sequencethat retains at least one structural or functional characteristic of thepolynucleotide. In one embodiment of the present invention,polynucleotide fragments are those that encode at least one(Gly-X-Y)_(n) region. Polynucleotide fragments can be of variablelength, for example, greater than 60 nucleotides in length, at least 100nucleotides in length, at least 1000 nucleotides in length, or at least10,000 nucleotides in length.

[0081] The phrase “percent similarity” (% similarity) refers to thepercentage of sequence similarity found in a comparison of two or morepolypeptide or polynucleotide sequences. Percent similarity can bedetermined by methods well-known in the art. For example, percentsimilarity between amino acid sequences can be calculated using theClustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The Clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentsimilarity can be calculated by other methods known in the art, forexample, by varying hybridization conditions, and can be calculatedelectronically using programs such as the MEGALIGN program (DNASTARInc., Madison, Wis.).

[0082] As used herein, the term “plant” includes reference to one ormore plants, i.e., any eukaryotic autotrophic organisms, such asangiosperms and gymnosperms, monotyledons and dicotyledons, etc.,including, but not limited to, soybean, cotton, alfalfa, flax, tomato,sugar, beet, sunflower, potato, tobacco, maize, wheat, rice, lettuce,banana, cassava, safflower, oilseed, rape, mustard, canola, hemp, algae,kelp, etc. The term “plant” also encompasses one or more plant cells.The term “plant cells” includes, but is not limited to, vegetativetissues and organs such as seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, tubers, corms, bulbs, flowers,fruits, cones, microspores, etc.

[0083] The term “post-translational enzyme” refers to any enzyme thatcatalyzes post-translational modification of, for example, any collagenor procollagen. The term encompasses, but is not limited to, forexample, prolyl hydroxylase, peptidyl prolyl isomerase, collagengalactosyl hydroxylysyl glucosyl transferase, hydroxylysyl galactosyltransferase, C-proteinase, N-proteinase, lysyl hydroxylase, and lysyloxidase.

[0084] As used herein, the term “promoter” generally refers to aregulatory region of nucleic acid sequence capable of initiating,directing, and mediating the transcription of a polynucleotide sequence.Promoters may additionally comprise recognition sequences, such asupstream or downstream promoter elements, which may influence thetranscription rate.

[0085] The term “non-constitutive promoters” refers to promoters thatinduce transcription via a specific tissue, or may be otherwise underenvironmental or developmental controls, and includes repressible andinducible promoters such as tissue-preferred, tissue-specific, and celltype-specific promoters. Such promoters include, but are not limited to,the AdH 1 promoter, inducible by hypoxia or cold stress, the Hsp70promoter, inducible by heat stress, and the PPDK promoter, inducible bylight.

[0086] Promoters which are “tissue-preferred” are promoters thatpreferentially initiate transcription in certain tissues. Promoterswhich are “tissue-specific” are promoters that initiate transcriptiononly in certain tissues. “Cell type-specific” promoters are promoterswhich primarily drive expression in certain cell types in at least oneorgan, for example, vascular cells.

[0087] “Inducible” or “repressible” promoters are those under control ofthe environment, such that transcription is effected, for example, by anenvironmental condition such as anaerobic conditions, the presence oflight, biotic stresses, etc., or in response to internal, chemical, orbiological signals, e.g., glyceraldehyde phosphate dehydrogenase, AOX 1and AOX2 methanol-inducible promoters, or to physical damage.

[0088] As used herein, the term “constitutive promoters” refers topromoters that initiate, direct, or mediate transcription, and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters, include, butare not limited to, the cauliflower mosaic virus (CaMv) 35S, the 1′- or2′- promoter derived from T-DNA of Agrobacteriuam tumefaciens, theubiquitin 1 promoter, the Smas promoter, the cinnamyl alcoholdehydrogenase promoter, glyceraldehyde dehydrogenase promoter, and theNos promoter, etc.

[0089] The term “purified” as it is used herein denotes that theindicated molecule is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. The term preferably contemplates that the molecule of interest ispresent in a solution or composition at least 80% by weight; preferably,at least 85% by weight; more preferably, at least 95% by weight; and,most preferably, at least 99.8% by weight. Water, buffers, and othersmall molecules, especially molecules having a molecular weight of lessthan about one kDa, can be present.

[0090] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0091] A “substitution” is the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

[0092] The term “transfection” as used herein refers to the process ofintroducing an expression vector into a cell. Various transfectiontechniques are known in the art, for example, microinjection,lipofection, or the use of a gene gun.

[0093] “Transformation”, as defined herein, describes a process by whichexogenous nucleic acid sequences, e.g., DNA, enters and changes arecipient cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the type of host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation, heatshock, lipofection, and particle bombardment. Such “transformed” cellsinclude stably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome, and also include cells which transiently expressthe inserted nucleic acid for limited periods of time.

[0094] As used herein, the term “vaccine” refers to a preparation ofkilled or modified microorganisms, living attenuated organisms, orliving fully virulent organisms, or any other agents, including, but notlimited to peptides, proteins, biological macromolecules, or nucleicacids, natural, synthetic, or semi-synthetic, administered to produce orartificially increase immunity to a particular disease, in order toprevent future infection with a similar entity. Vaccines can containlive or inactivated microorganisms or agents, including viruses andbacteria, as well as subunit, synthetic, semi-synthetic, or recombinantDNA-based.

[0095] Vaccines can be monovalent (a single strain/microorganism/diseasevaccine) consisting of one microorganism or agent (e.g., poliovirusvaccine) or the antigens of one microorganism or agent. Vaccines canalso be multivalent, e.g., divalent, trivalent, etc. (a combinedvaccine), consisting of more than one microorganism or agent (e.g., ameasles-mumps-rubella (MMR) vaccine) or the antigens of more than onemicroorganism or agent.

[0096] Live vaccines are prepared from living microorganisms. Attenuatedvaccines are live vaccines prepared from microorganisms which haveundergone physical alteration (such as radiation or temperatureconditioning) or serial passage in laboratory animal hosts or infectedtissue/cell cultures, such treatments producing avirulent strains orstrains of reduced virulence, but maintaining the capability of inducingprotective immunity. Examples of live attenuated vaccines includemeasles, mumps, rubella, and canine distemper. Inactivated vaccines arevaccines in which the infectious microbial components have beendestroyed, e.g., by chemical or physical treatment (such as formalin,beta-propiolactone, or gamma radiation), without affecting theantigenicity or immunogenicity of the viral coat or bacterial outermembrane proteins. Examples of inactivated or subunit vaccines includeinfluenza, Hepatitis A, and poliomyelitis (IPV) vaccines.

[0097] Subunit vaccines are composed of key macromolecules from, e.g.,the viral, bacterial, or other agent responsible for eliciting an immuneresponse. These components can be obtained in a number of ways, forexample, through purification from microorganisms, generation usingrecombinant DNA technology, etc. Subunit vaccines can contain syntheticmimics of any infective agent. Subunit vaccines can includemacromolecules such as bacterial protein toxins (e.g., tetanus,diphtheria), viral proteins (e.g., from influenza virus),polysaccharides from encapsulated bacteria (e.g., from Haemophilusinfluenzae and Streptococcus pneumonia), and viruslike particlesproduced by recombinant DNA technology (e.g., hepatitis B surfaceantigen), etc.

[0098] Synthetic vaccines are vaccines made up of small syntheticpeptides that mimic the surface antigens of pathogens and areimmunogenic, or may be vaccines manufactured with the aid of recombinantDNA techniques, including whole viruses whose nucleic acids have beenmodified.

[0099] Semi-synthetic vaccines, or conjugate vaccines, consist ofpolysaccharide antigens from microorganisms attached to protein carriermolecules.

[0100] DNA vaccines contain recombinant DNA vectors encoding antigens,which, upon expression of the encoded antigen in host cells having takenup the DNA, induce humoral and cellular immune responses against theencoded antigens.

[0101] Vaccines have been developed for a variety of infectious agents.The present invention is directed to recombinant gelatins that can beused in vaccine formulations regardless of the agent involved, and arethus not limited to use in the vaccines specifically described herein byway of example. Vaccines include, but are not limited to, vaccines forvacinnia virus (small pox), polio virus (Salk and Sabin), mumps,measles, rubella, diphtheria, tetanus, Varicella-Zoster (chickenpox/shingles), pertussis (whopping cough), Bacille Calmette-Guerin (BCG,tuberculosis), haemophilus influenzae meningitis, rabies, cholera,Japanese encephalitis virus, salmonella typhi, shigella, hepatitis A,hepatitis B, adenovirus, yellow fever, foot-and-mouth disease, herpessimplex virus, respiratory syncytial virus, rotavirus, Dengue, West Nilevirus, Turkey herpes virus (Marek's Disease), influenza, and anthrax.The term vaccine as used herein includes reference to vaccines tovarious infectious and autoimmune diseases and cancers that have been orthat will be developed, for example, vaccines to various infectious andautoimmune diseases and cancers, e.g., vaccines to HIV, HCV, malaria,and vaccines to breast, lung, colon, renal, bladder, and ovariancancers.

[0102] A polypeptide or amino acid “variant” is an amino acid sequencethat is altered by one or more amino acids from a particular amino acidsequence. A polypeptide variant may have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties tothe amino acid replaced, e.g., replacement of leucine with isoleucine. Avariant may also have nonconservative changes, in which the substitutedamino acid has physical properties different from those of the replacedamino acid, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Preferably, amino acid variants retain certain structural orfunctional characteristics of a particular polypeptide. Guidance indetermining which amino acid residues may be substituted, inserted, ordeleted may be found, for example, using computer programs well known inthe art, such as LASERGENE software (DNASTAR Inc., Madison, Wis.).

[0103] A polynucleotide variant is a variant of a particularpolynucleotide sequence that preferably has at least about 80%, morepreferably at least about 90%, and most preferably at least about 95%polynucleotide sequence similarity to the particular polynucleotidesequence. It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of variantpolynucleotide sequences encoding a particular protein, some bearingminimal homology to the polynucleotide sequences of any known andnaturally occurring gene, may be produced. Thus, the inventioncontemplates each and every possible variation of polynucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard codon triplet genetic code, and all such variations are to beconsidered as being specifically disclosed.

Invention

[0104] The present invention provides recombinant gelatins and methodsfor producing these gelatins. The recombinant gelatins of the presentinvention provide reproducible and improved performance, and answervarious health and other concerns. Using the present methods, gelatincan be directly manufactured, rather than extracted from animal sourcesthrough lengthy and harsh processes. The recombinant gelatin of thepresent invention is free of pathogens, for example, pathogenicbacteria, transmissible spongiform encephalopathies (TSEs), etc. Thepresent methods minimize variability and allow for a degree ofreproducibility unattainable in current extraction methods.

[0105] Safety issues, such as concern over potential immunogenic, e.g.,antigenic and allergenic, responses, have arisen regarding the use ofanimal-derived products. The inability to completely characterize,purify, or reproduce animal-source gelatin mixtures used currently is ofongoing concern in the pharmaceutical and medical communities.Additional safety concerns exist with respect to bacterial contaminationand endotoxin loads resulting from the extraction and purificationprocesses.

[0106] The recombinant gelatins of the present invention address theseconcerns as they are virtually free of bacterial contamination orendotoxins. Furthermore, the recombinant human gelatins of the presentinvention will offer distinct advantages over animal-derivedcounterparts currently in use, as the use of gelatins derived fromnative human sequence can eliminate the risk of immune response due tothe use of non-human, animal-derived proteins.

[0107] In addition, the present gelatins can be produced as various anddistinct materials, with characteristics optimized for particularapplications. The resultant products are internally more consistent anduniform than are currently available gelatins derived from animalsources.

[0108] In one embodiment, the present invention provides a recombinantgelatin. The gelatin can be produced using sequences from variousspecies including, but not limited to, human, bovine, porcine, equine,and piscine species. The gelatin of the present invention has increasedpurity as compared to the gelatin products of current methods ofmanufacture, and has a reduced protein load and reduced levels ofendotoxins and other contaminants, including nucleic acids,polysaccharides, prions, etc. The present gelatin is thus safer to usethan gelatin manufactured by current methods, and can be administered toor ingested by humans and animals at a higher dosage while minimizingthe risk of negative side effects.

[0109] The gelatins of the present invention have increased activity andworkability compared to commercial gelatins, as the present gelatin canbe produced directly with characteristics optimized for specific uses,improving one's ability to use and formulate the gelatin. While gelatinscurrently extracted from animal sources are heterogeneous products witha wide range in molecular weights throughout a given batch or sample,the gelatins of the present invention include consistent, homogeneous,and reproducible products.

[0110] The recombinant gelatins of the present invention can be producedin a variety of methods. In one method, the recombinant gelatin isproduced through processing of recombinant collagen. (See, e.g.,Examples 7, 10, and 11.) In another method, the recombinant gelatin isproduced directly from the expression of altered collagen constructs,i.e., constructs containing a polynucleotide encoding at least onecollagenous domain, but not encoding naturally occurring collagen. (See,e.g., Examples 1, 4, and 6.) In another aspect, the recombinant gelatinis derived from polypeptides which are not full-length naturallyoccurring collagen or procollagen, but which contain at least onecollagenous domain. (See, e.g., SEQ ID NOs:15 through 25, 30, 31, and33.) Recombinant gelatins can also comprise sequences containingadditional N-terminal or C-terminal propeptides. (See, e.g., SEQ IDNOs:26 through 29.)

[0111] In one aspect, the recombinant gelatin of the present inventionis derived from recombinant collagens or procollagens. Collagenmolecules generally result from trimeric assembly of polypeptide chainscontaining (Gly-X-Y-)_(n) repeats which allow for the formation oftriple helical domains under normal biological conditions. (See, e.g.,van der Rest et al., (1991), FASEB J. 5:2814-2823.) At present, abouttwenty distinct collagen types have been identified in vertebrates,including bovine, ovine, porcine, chicken and human collagens. Adetailed description of structure and biological functions of thevarious types of naturally occurring collagens can be found, forexample, in Ayad et al., The Extracellular Matrix Facts Book, AcademicPress, San Diego, Calif.; Burgeson, R. E., and Nimmi (1992) “Collagentypes: Molecular Structure and Tissue Distribution,” Clin. Orthop.282:250-272; Kielty, C. M. et al. (1993) “The Collagen Family:Structure, Assembly And Organization In The Extracellular Matrix,” inConnective Tissue And Its Heritable Disorders, Molecular Genetics, AndMedical Aspects, Royce, P. M. and Steinmann, B., Eds., Wiley-Liss, NY,pp. 103-147; and Prockop and Kivirikko (1995) “Collagens: Molecularbiology, diseases, and potentials for therapy”, Annu Rev Biochem64:403-434.

[0112] Type I collagen is the major fibrillar collagen of bone and skin,comprising approximately 80-90% of an organism's total collagen. Type Icollagen is the major structural macromolecule present in theextracellular matrix of multicellular organisms and comprisesapproximately 20% of total protein mass. Type I collagen is aheterotrimeric molecule comprising two α1(I) chains and one α2(I) chain,which are encoded by the COL1A1 and COL1A2 genes, respectively. Othercollagen types are less abundant than type I collagen and exhibitdifferent distribution patterns. For example, type II collagen is thepredominant collagen in cartilage and vitreous humor, while type IIIcollagen is found at high levels in blood vessels and to a lesser extentin skin.

[0113] Type III collagen is a major fibrillar collagen found in skin andvascular tissues. Type III collagen is a homotrimeric collagencomprising three identical α1(III) chains encoded by the COL3A1 gene.Methods for purifying various collagens from tissues can be found, forexample, in, Byers et al. (1974) Biochemistry 13:5243-5248; and Millerand Rhodes (1982) Methods in Enzymology 82:33-64.

[0114] Post-translational enzymes are important to the biosynthesis ofprocollagens and collagens. For example, prolyl 4-hydroxylase is apost-translational enzyme necessary for the synthesis of procollagen orcollagen by cells. This enzyme hydroxylates prolyl residues in theY-position of repeating Gly-X-Y sequences to 4-hydroxyproline. (See,e.g., Prockop et al. (1984) N. Engl. J. Med. 311:376-386.) Unless anappropriate number of Y-position prolyl residues are hydroxylated to4-hydroxyproline by prolyl 4-hydroxylase, the newly synthesized chainscannot maintain a stable triple-helical conformation. Moreover, if nohydroxylation or under-hydroxylation occurs, the polypeptides are notsecreted properly and may be degenerated.

[0115] Vertebrate prolyl 4-hydroxylase is an α₂ β₂ tetramer. (See, e.g.Berg and Prockop (1973) J. Biol. Chem. 248:1175-1192; and Tuderman etal. (1975) Eur. J. Biochem. 52:9-16.) The α subunits contain thecatalytic sites involved in the hydroxylation of prolyl residues, butare insoluble in the absence of β subunits. The β subunits, proteindisulfide isomerases, catalyze thiol/disulfide interchanges, leading toformation of disulfide bonds essential to establishing a stable protein.The β subunits retain 50% of protein disulfide isomerase activity whenpart of the prolyl 4-hydroxylase tetramer. (See, e.g., Pihlajaniemi etal. (1987) Embo J. 6:643-649; Parkkonen et al. (1988) Biochem. J.256:1005-1011; and Koivu et al. (1987) J. Biol. Chem. 262:6447-6449.)

[0116] Active recombinant human prolyl 4-hydroxylase has been producedin, e.g., Sf9 insect cells and in yeast cells, by simultaneouslyexpressing the α and β subunits. (See, e.g., Vuori et al. (1992) Proc.Natl. Acad. Sci. USA 89:7467-7470; U.S. Pat. No. 5, 593,859.) Inaddition to prolyl 4-hydroxylase, other collagen post-translationalenzymes have been identified and reported in the literature, includingC-proteinase, N-proteinase, lysyl oxidase, lysyl hydroxylase, etc. (See,e.g., Olsen et al. (1991) Cell Biology of Extracellular Matrix, 2^(nd)ed., Hay editor, Plenum Press, New York.)

[0117] The present invention specifically contemplates the use of anycompound, biological or chemical, that confers hydroxylation, e.g.,proline hydroxylation and/or lysyl hydroxylation, etc., as desired, tothe present recombinant gelatins. This includes, for example, prolyl4-hydroxylase from any species, endogenously or exogenously supplied,including various isoforms of prolyl 4-hydroxylase and any variants orfragments or subunits of prolyl 4-hydroxylase having the desiredactivity, whether native, synthetic, or semi-synthetic, and otherhydroxylases such as prolyl 3-hydroxylase, etc. (See, e.g., U.S. Pat.No. 5,928,922, incorporated by reference herein in its entirety.) In oneembodiment, the prolyl hydroxylase activity is conferred by a prolylhydroxylase derived from the same species as the polynucleotide encodingrecombinant gelatin or encoding a polypeptide from which recombinantgelatin can be derived. In a further embodiment, the prolyl4-hydroxylase is human and the encoding polynucleotide is derived fromhuman sequence.

[0118] The present invention provides methods for manipulating thethermoplasticity of gelatin in order to produce a material with thedesired physical characteristics. In one method, the encodingpolynucleotides are expressed in a host system having endogenous prolylhydroxylase or alternate hydroxylases, such as certain mammalian orinsect cells, or transgenic animals, or plants or plant cells. In such asystem, the present invention provides methods for producing a mixtureof recombinant gelatins having a range of percentages of hydroxylation,i.e., non-hydroxylated, partially hydroxylated, and fully hydroxylatedportions. For example, in one method of producing recombinant gelatinswith varying percentages of hydroxylation, the hydroxylation isconferred by endogenous prolyl hydroxylase in, e.g., a transgenicanimal, and the distribution of percentage hydroxylation ranges fromnon-hydroxylated to fully hydroxylated, and the melting temperatures ofthe material produced range from 28° C. to 36° C., with a median T_(m)value of around 30° C. to 32° C. If desired, different fractions of thematerial can be isolated along a temperature gradient, as might benecessary if downstream uses require selecting, for example, the morefully hydroxylated materials, such as those sufficiently hydroxylated toretain triple helical structure at, e.g., body temperature (37° C.).

[0119] In another embodiment, recombinant gelatins are produced in asystem, e.g., a transgenic animal, in which hydroxylation issupplemented with exogenous prolyl hydroxylase. In one aspect, such amethod of producing recombinant gelatins provides recombinant gelatinsranging from non-hydroxylated to fully hydroxylated. The fraction ofrecombinant gelatins more fully hydroxylated will be substantiallylarger in recombinant material produced in the presence of exogenousprolyl hydroxylase than in recombinant material produced only in thepresence of endogenous prolyl hydroxylase. Therefore, the meltingtemperatures of the material produced can range from, for example, 28°C. to 40° C., having a median T_(m) value of around 34° C. to 36° C.Such a gelatin mixture could be appropriate for use in a variety ofapplications, such as gel capsule manufacture, without requiring anyfractionation or separation of differently hydroxylated portions.

[0120] The above methods provide for production of recombinant materialswith a range of melting temperatures, that can be easily divided, forexample, using a temperature gradient to separate materials solid at aparticular temperature, e.g., 36° C., from those liquid at a particulartemperature. Furthermore, the present invention provide forcost-effective methods of producing a material which, withoutseparation, is suitable for use in bulk applications. For example, themanufacture of gel capsules could involve the use of recombinant gelatinproduced by the above methods, wherein the recombinant material, havinga range of melting temperatures, had a desirable melting temperature ofaround 33° C., such gelatin melting at body temperatures, and thus beingsuitable for swallowing and digestion. In the present methods, therecombinant gelatin can be produced directly in the desired system,e.g., a transgenic animal, or can be derived, for example, throughhydrolysis, e.g., acid, thermal, or enzymatic, from recombinantcollagens produced in the desired system.

[0121] In one embodiment, the present invention provides a method ofproducing recombinant gelatin comprising producing recombinant collagenand deriving recombinant gelatin from the recombinant collagen. In oneaspect, the method comprises the expression of at least onepolynucleotide sequence encoding a collagen or procollagen, or fragmentor variant thereof, and at least one polynucleotide encoding a collagenpost-translational enzyme or a subunit thereof. (See, e.g., U.S. Pat.No. 5,593,859, incorporated by reference herein in its entirety.) Thepresent recombinant gelatins can be derived from recombinant collagensusing procedures known in the art. (See, e.g., Veis (1965) Int RevConnect Tissue Res, 3:113-200.) For example, a common feature of allcollagen-to-gelatin extraction processes is the loss of the secondarystructure of the collagen protein, and in the majority of instances, analteration in collagen structure. The collagens used in producing thegelatins of the present invention can be processed using differentprocedures depending on the type of gelatin desired.

[0122] Gelatin of the present invention can be derived fromrecombinantly produced collagen, or procollagens or other collagenouspolypeptides, or from cell cultures, e.g., vertebrate cell cultures, bya variety of methods known in the art. For example, gelatin may bederived directly from the cell mass or the culture medium by takingadvantage of gelatin's solubility at elevated temperatures and itsstability under conditions of low or high pH, low or high saltconcentrations, and high temperatures. Methods, processes, andtechniques of producing gelatin compositions from collagen includedigestion with proteolytic enzymes at elevated temperatures, denaturingthe triple helical structure of the collagen utilizing detergents, heat,or various denaturing agents well known in the art, etc. In addition,various steps involved in the extraction of gelatin from animal orslaughterhouse sources, including treatment with lime or acids, heatextraction in aqueous solution, ion exchange chromatography, cross-flowfiltration, and various methods of drying can be used to derive thegelatin of the present invention from recombinant collagen.

[0123] In one aspect, the gelatin of the present invention is comprisedof denatured triple helices, and comprises at least one collagensubunit, collagen chain, or fragment thereof. The Gly-X-Y units within aparticular collagen chain, subunit, or fragment thereof may be the sameor different. Preferably, X and Y are either proline or hydroxyproline,and glycine appears in about every third residue position of thecomponent chain. The amino acids of X and Y are proline orhydroxyproline, and each Gly-X-Y unit is the same or different. Inanother embodiment, the recombinant gelatin of the present inventioncomprises an amino acid sequence of (Gly-X-Y)_(n) wherein X and Y areany amino acid.

[0124] In one embodiment, the present gelatin is derived from arecombinant collagen of one type that is substantially free fromcollagen of any other collagen type. In one aspect, the recombinantcollagen is type I collagen. In another aspect, the recombinant collagenis type III collagen. In another embodiment of the present invention,the recombinant collagen is human recombinant collagen. Furtherembodiments of the invention, in which the recombinant collagen is ofany one collagen type, such as any one of collagen types I through XX,inclusively, or any other collagen, natural, synthetic, orsemi-synthetic, are specifically contemplated. Embodiments in which therecombinant gelatin is derived from specified mixtures of any one ormore of any of collagen types I through XX, inclusively, or any othercollagen, natural, synthetic, or semi-synthetic, are specificallycontemplated.

[0125] The present methods of producing recombinant gelatin have anumber of advantages over traditional methods of gelatin extraction.Most importantly, the present methods provide a reliable non-tissuesource of gelatin containing native collagen sequence. In addition,current methods of extraction do not allow for any natural source ofhuman gelatin, such as might be advantageous for use in various medicalapplications. The present invention specifically provides recombinantgelatins derived from human sequences, compositions comprisingrecombinant human gelatins, and methods of producing these gelatins. Therecombinant human gelatin is non-immunogenic as applied inpharmaceutical and medical processes, and various uses thereof are alsocontemplated.

[0126] In another aspect, the present invention provides for theproduction of the present gelatin from engineered constructs capable ofexpressing gelatin in various forms. This invention specificallycontemplates methods of producing gelatin using recombinant prolylhydroxylase and various synthetic constructs, including non-nativecollagen constructs. Further, the present invention provides recombinantgelatins that can be designed to possess the specific characteristicsneeded for a particular application. Methods for producing thesegelatins are also contemplated. Using the current methods, one couldproduce a gelatin with the desired gel strength, viscosity, meltingcharacteristics, isoelectric profile, pH, degree of hydroxylation,amino-acid composition, odor, color, etc. In one method according to thepresent invention, non-hydrolyzed gelatin is produced, and can besubsequently hydrolyzed fully or partially, if desired.

Properties of Gelatin

[0127] The various physical properties of gelatin define its usefulnessin particular applications. Gelatin provides unique performance basedon, for example, its amphoteric nature, its ability to formthermo-reversible gels, its protective colloidal and surface activeproperties, and its contribution to viscosity and stability. In a numberof applications, gelatin is used, for example, as an emulsifier,thickener, or stabilizer; as an agent for film or coating formation; asa binding agent; as an adhesive or glue; or as a flocculating agent.

[0128] Raw materials, types of pre-treatment, and extraction processesall effect the composition of gelatin polypeptides obtained duringconventional manufacture. Currently available animal products are thusheterogeneous protein mixtures of polypeptide chains. Gelatin moleculescan be fairly large, with the molecular weight within a particularsample ranging from a few to several hundred kDa. The molecular weightdistribution of gelatin in a particular lot can be critical, as weightdistribution can influence, for example, the viscosity and/or gelstrength of a gelatin sample.

[0129] In general, the viscosity of a gelatin solution increases withincreasing concentration and with decreasing temperature. A higherviscosity solution would be preferred, for example, for gelatin used asa stabilizer or thickener. In some applications, liquid gelatins arepreferred, such as in various emulsifying fluids, etc. Viscosity of agelatin solution increases with increasing molecular weight of thegelatin components. A high-viscosity gelatin solution could consist,therefore, of a high concentration of low molecular weight gelatins, orof a lower concentration of high molecular weight gelatins. Viscosityalso affects gel properties including setting and melting point.High-viscosity gelatin solutions provide gels with higher melting andsetting rates than do lower viscosity gelatin solutions.

[0130] The thermoreversibility and thermoplasticity of gelatin areproperties exploited in a number of applications, for example, in themanufacture of gel capsules and tablets. Gelatin can be heated, moldedor shaped as appropriate, and cooled to form a capsule or tablet coatingthat has unique properties at homeostatic temperatures. The gelatin willbegin to melt at mouth temperature, easing swallowing, and become liquidat body temperatures.

[0131] Gelatins of various gel strengths are suitable for use indifferent applications. The firmness or strength of the set gel istypically measured by calculating the Bloom value, which can bedetermined using international standards and methodology. Briefly, theBloom strength is a measurement of the strength of a gel formed by a6.67% solution of gelatin in a constant temperature bath over 18 hours.A standard Texture Analyzer is used to measure the weight in gramsrequired to depress a standard AOAC (Association of OfficialAgricultural Chemists) plunger 4 millimeters into the gel. If the weightin grams required for depression of the plunger is 200 grams, theparticular gelatin has a Bloom value of 200. (See, e.g., United StatesPharmacopoeia and Official Methods of Analysis of AOAC International,17^(th) edition, Volume II.)

[0132] Commercial gelatins can thus be graded and sold on Bloomstrength. Different ranges of Bloom values are appropriate for differentuses of gelatin; for example, gelatins for use in various industrialapplications, e.g., concrete stabilization, sand casting, molds, glues,coatings, etc., will be selected from a wide range of varying Bloomstrengths, depending on the performance characteristics desired.Gelatins with varying Bloom strengths are also desired in themanufacture of various pharmaceutical products. For example, soft gelcapsules are typically manufactured using ossein or skin gelatin with aBloom value of about 150 to 175 and/or porcine-derived gelatin with aBloom value of about 190 to 210, or blends thereof, while hard gelcapsules might use a gelatin with a Bloom value of about 220 to 260. Infood applications, gelatin used, for example, as a thickener inmarshmallows or other confectionary products might have a Bloom strengthof around 250. Various applications, including certain emulsifyingfluids in photographic applications, and various industrial coatings,involve the use of non-gelling gelatins.

[0133] The present invention provides for the production of recombinantgelatins with different Bloom strengths. In one aspect, the presentinvention provides, for example, for the manufacture of gelatins withBloom strengths of around 50, 100, 150, 200, 250, and 300. In oneembodiment, the present invention provides for the production of arecombinant gelatin having a Bloom strength of around 400. Such agelatin can be used, for example, in the manufacture of gel capsules,and could allow for the manufacture of a lighter and thinner capsule, asless material would need to be used to provide a gel of sufficientstrength. Recombinant gelatins with Bloom strengths of under 100, andfrom 0 to 100, inclusively, are also contemplated.

[0134] The present invention provides methods for designing recombinantgelatins with the physical properties desired for particularapplications. In one embodiment, the present invention providesrecombinant gelatins comprising uniform molecules of a specifiedmolecular weight or range of molecular weights, and methods forproducing these recombinant gelatins. Such homogeneous and uniformmaterials are advantageous in that they provide a reliable source ofproduct with predictable performance, minimizing variability in productperformance and in manufacturing parameters. Currently, gelatin fromdifferent lots must sometimes be blended in order to produce a mixturewith the desired physical characteristics, such as the viscosity or gelstrength, etc., provided by a particular molecular weight or molecularweight range.

[0135] In applications in which a specific molecular weight range ofrecombinant gelatin would be preferred to a recombinant gelatin with aspecific molecular weight, the present invention provides suchmaterials. Using the recombinant gelatins of the present invention, amanufacturer could, for example, mix recombinant gelatins from lots withspecified molecular weights, in certain percentages, in order to achievea mixture with the desired molecular weight range. Additionally, thepresent recombinant gelatins are inherently more uniform and of greaterconsistency than currently available commercial products. In one methodof the present invention, recombinant collagen is processed, such as byacid or heat hydrolysis, to produce recombinant gelatin of a molecularweight range narrower than that of currently available gelatin products.Using suitable and controllable hydrolysis conditions, the presentmethods produced recombinant human gelatins with molecular weightdistributions similar to those of commercially available gelatins, aswell as recombinant gelatins with ranges narrower than those of themolecular weight ranges of currently available products. (See Examples 9and 10.)

[0136] The present invention provides recombinant gelatins of uniformmolecular weight or specified ranges of molecular weights, removingvariability and unpredictability, and allowing for fine-tuning ofprocesses and predictable behavior. The present methods allow fro theproduction of recombinant gelatins of any desired molecular weight orrange of molecular weights. For example, in one embodiment, therecombinant gelatin has a molecular weight greater than 300 kDa. Inanother embodiment, the recombinant gelatin has a molecular weight rangeof from about 150 to 250 kDa, or of from about 250 to 350 kDa. Othermolecular weight ranges are specifically contemplated, including, butnot limited to, the following molecular weight ranges: about 0 to 50kDa, about 50 to 100 kDa, about 100 to 150 kDa, about 150 to 200 kDa,about 200 to 250 kDa, about 250 to 300 kDa, and about 300 to 350 kDa.

[0137] In another aspect, recombinant gelatin with a molecular weightsimilar to that of some commercially available gelatins, of from about10 to 70 kDa, could be produced. In preferred embodiments, the presentinvention provides recombinant gelatins narrower molecular weightranges, not currently available in commercial products, such as fromabout 10 to 30 kDa, about 30 to 50 kDa, and about 50 to 70 kDa. In aparticular embodiment, a recombinant gelatin with a chain lengthconferring specific properties appropriate to the intended applicationis provided. In various embodiments of the present invention,recombinant gelatins with uniform molecular weights of approximately 1kDa, 5 kDa, 8 kDa, 9 kDa, 14 kDa, 16 kDa, 22 kDa, 23 kDa, and 44 kDa arecontemplated. (See, e.g., Table 2.)

[0138] In particular, in one method of the present invention, gelatin isproduced from shortened collagen sequences, for example, the sequencesidentified in Table 2. These sequences represent specific collagenousdomains and encode short forms of gelatin.

[0139] The present gelatins are capable of retaining valuable physicalcharacteristics of gelatin, for example, film-forming abilities, whilepossessing average molecular weights lower or higher than those ofconventionally derived animal gelatin. Various modifications of collagensequences, including, for example, denaturing of the collagen, collagenchain, subunit, or fragments thereof, or varying degrees ofhydroxylation, can be made that will produce gelatin with specificphysical properties, i.e., a higher or lower melting point thanconventional gelatin, different amino acid compositions, specificmolecular weights or ranges of molecular weights, etc., and suchvariations are specifically contemplated herein.

[0140] The molecular weight of a typical fibril-forming collagenmolecule, such as type I collagen, is 300 kDa. In some applications,such as those in which high molecular weight gelatins are used, it mightbe desirable to produce a gelatin with a greater molecular weight thanthat of currently available extracted gelatin. Therefore, in oneembodiment of the present invention, gelatin can be produced containingmolecules larger than the collagen from which commercial gelatin iscurrently extracted. The resultant higher molecular weight gelatinproduct can be used directly in various applications in which itsphysical properties would be desirable, or can be divided andsubsequently treated to produce molecules of a smaller sizes.

[0141] In one embodiment, gelatin can be produced using collagens largerthan those available in conventional animal sources. For example, thepresent methods of production could be adapted to produce theacid-soluble cuticle collagens derived from the body walls ofvestimentiferan tube worm Riftia pachyptila (molecular weight ˜2600 kDa)and annelid Alvinella pompejana (molecular weight ˜1700 kDa). Thesecollagens could be adapted to the present methods of production toproduce larger molecules than those from which currently availablegelatin is extracted, and the resultant product could be treated toproduce gelatins as desired.

[0142] It is specifically contemplated that gelatins of variousmolecular weights can be produce by a variety of methods according tothe present invention. For example, characteristics of the presentrecombinant gelatins, e.g., percentage hydroxylation, degrees ofcross-linking, etc., can be varied to produce recombinant gelatins withthe desired molecular weights. In one aspect, for example, the presentinvention provides a method for producing large molecular weightrecombinant gelatins by using cross-linking agents known in the art tocross-link gelatin polypeptides. (See discussion, infra.)

[0143] In another aspect of the present invention, polypeptides fromwhich gelatins could be derived are expressed from engineered constructscontaining multiple copies of all or fragments of native collagensequence. For example, in one embodiment, the present invention providesan altered collagen construct comprising multiple copies of thecollagenous domain of type I collagen. In another embodiment, theconstruct comprises multiple copies of the collagenous domain of typeIII collagen. In a further embodiment, the construct comprises copies oftype I and type III collagenous domains. The present invention providesfor the use of single or multiple copies of all or portions of sequencesencoding any collagen, including collagens type I through XX, inclusive.It is specifically contemplated that the present methods allow for theproduction of gelatins derived from more than one type of collagen. Inone embodiment, recombinant gelatins derived from more than one type ofcollagen are co-expressed in an expression system, e.g., a host cell,transgenic animal, etc., such that a mixture of gelatins is produced.

[0144] In another embodiment, the present invention provides a methodfor producing gelatin without derivation from a collagen or procollagentriple helical stage. In one aspect, this involves production ofrecombinant gelatin by expression of various constructs in ahigh-temperature expression system, such as one relying on thermophilicorganisms, that does not allow the formation of triple helicalstructures, but permits the activity of prolyl hydroxylase. The presentgelatin could also be derived from collagen constructs containingmutations, additions, or deletions that prevent triple helicalformation. In another aspect, this involves production of gelatin fromshortened constructs that do not allow for formation of triple helicesat regular temperatures, i.e., 37° C. Alternatively, gelatin can beproduced in the presence of inhibitors of triple helix formation, forexample, polyanions, that are co-expressed with the biosyntheticcollagen constructs. Additionally, the biosynthetic gelatin of thepresent invention could be derived from recombinantly produced collagenchains that do not form triple helices.

[0145] In another embodiment, the invention provides a method ofderiving gelatin from non-hydroxylated collagen or collagen in whichthere is partial rather than full hydroxylation of proline residues. Inone aspect, this method comprises deriving gelatin from collagenexpressed in the absence of prolyl hydroxylase, for example, in aninsect expression system without prolyl hydroxylase. (See, e.g.,Myllyharju et al. (1997) J. Biol. Chem. 272, 21824-21830.) In one methodaccording to the present invention, gelatin is derived from thepartially hydroxylated or non-hydroxylated collagen. Hydroxylation canbe conferred, for example, by in vitro administration of hydroxylases.In one method, a low degree of substitution of hydroxyproline forproline can be forced by providing hydroxyproline to, e.g., bacterial oryeast host cells.

[0146] The present invention comprises fully hydroxylated, partiallyhydroxylated, and non-hydroxylated recombinant gelatins. In anotherembodiment, the method of the present invention comprises producing agelatin or gelatin precursor having a specific degree of hydroxylation.In a further aspect, the invention relates to a method of producinggelatin having from 20 to 80 percent hydroxylation, preferably, fromabout 30 to 60 percent hydroxylation, and, most preferably, about 40percent hydroxylation. (See Examples 4 and 5.) The partiallyhydroxylated recombinant gelatins of the present invention can beobtained through mixing specified percentages of recombinant gelatinswith different degrees of hydroxylation, or can be obtained directly.(See Examples 4 and 5.) Further, the invention provides methods forachieving partial hydroxylation of recombinant gelatins by administeringprolyl hydroxylase to non-hydroxylated recombinant gelatins in vitro,and controlling the length of the reaction.

[0147] There are limits to the extent to which the thermalcharacteristics of currently available animal-source gelatins can bealtered. The present invention specifically provides for methods ofproducing recombinant gelatin, wherein the recombinant gelatin has thespecific thermal characteristics desired for a particular application.Using the methods of the present invention, for example, the meltingpoint and/or gel strength of the recombinant gelatin can be manipulatedin a variety of ways. The temperature stability and/or gel strength ofrecombinant gelatin can be measured by a variety of techniqueswell-known in the art.

[0148] Generally, the melting point of gelatin increases as the degreeof hydroxylation increases. Using the methods of the present invention,it is possible to produce high molecular weight gelatins that, due tomanipulation of hydroxylation and/or cross-linking, etc., have a lowergel strength and/or lower melting point than those of currentlyavailable animal-source gelatins. Therefore, the present inventionprovides a recombinant gelatin with properties unattainable in variouscommercial products, suitable for use in applications where a highermolecular weight gelatin is desired, in order to provide increased filmstrength, etc., but a non-gelling or low strength gel product isdesired. In one embodiment, the present invention provides recombinantgelatin that has lower temperature stability due to incompletehydroxylation of proline residues.

[0149] Such a recombinant gelatin could be useful in a variety ofapplications. In gelatin produced by current extraction methods, onlyfish gelatin provides a high average molecular weight film-formingprotein that is non-gelling. The non-gelling and cold water-solubilitycharacteristics offered by non-gelling fish gelatin can be matched bycurrently available hydrolyzed bovine and porcine gelatins, but withcorresponding loss of film strength and flexibility, as the hydrolyzedgelatins are of lower average molecular weight. Therefore, in oneembodiment, the present invention provides a partially hydroxylatedrecombinant gelatin with lower gel strength and higher molecular weightthan that provided by currently available animal-source materials.

[0150] A higher molecular weight, lower gel strength recombinant gelatincould also be useful in various pharmaceutical applications, in whichstability is desired, but non- or low-gelling properties are desired inorder to maintain the malleability and integrity of the pharmaceuticalproduct. Such a recombinant gelatin could be used, for example, as aplasma expander, as its molecular weight could provide stability,increasing the residence time in circulation, and the altered settingpoint would prevent the material from gelling at room temperature,allowing the expander to be administered without warming. In oneembodiment, the present invention provides a partially hydroxylatedrecombinant gelatin suitable for use in pharmaceutical applications, forexample, as a plasma expander.

[0151] In another aspect, partially hydroxylated recombinant gelatin isobtained through expression of recombinant gelatin, or expression ofpolypeptides from which the present recombinant gelatin can be derived,in the absence of prolyl hydroxylase, for example, in an insectexpression system without prolyl hydroxylase. (See, e.g., Myllyharju etal. (1997) J. Biol. Chem. 272, 21824-21830.) Hydroxylation can occur atthe time of production or can be subsequently imposed through, e.g., invitro biological or chemical modification. In one method of the presentinvention, recombinant gelatins are derived from partially hydroxylatedor from fully hydroxylated collagen.

[0152] Gelatins derived from natural sources by currently availablemethods are greatly strengthened by the existence of covalentcross-links between lysine residues of the constituent collagenmolecules. Cross-linking occurs naturally in the extracellular spacefollowing collagen secretion and fibril formation, as prior tosecretion, certain lysine residues are hydroxylated by the enzyme lysylhydroxylase. The extracellular enzyme lysyl oxidase subsequentlydeamidates certain lysine and hydroxylysine residues in the collagenmolecules, yielding highly reactive aldehyde groups that reactspontaneously to form covalent bonds. The resulting cross-linkedcollagens yield gelatins of increased gel strength and increasedviscosity. Specifically, a higher degree of cross-linking results ingelatins with higher melting temperatures and greater gel strength.

[0153] In one aspect, the present invention provides recombinantgelatins that are cross-linked, resulting in higher molecular weightgelatins. (See Example 7.) Cross-linking can be imposed by differentmethods, such as by biological or chemical modification. For example, inone embodiment, recombinant gelatin or a polypeptide from which gelatincan be derived is expressed in the presence of lysyl hydroxylase andlysyl oxidase. In another embodiment, the polypeptide is modified bycross-linking after expression. In a further aspect, the presentinvention provides for imposition of cross-linking by chemical means,such as by reactive chemical cross-linkers, for example 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC). (SeeExample 7.) Other chemical cross-linking agents, such asbis(sulfosuccinimidyl) suberate (BS³), 3,3′-dithiobis(sulfosuccinimidyl)propionate (DTSSP), and Tris-sulfosuccinimidyl aminotnacetate(Sulfo-TSAT) may also be used, as can various agents known in the art.Additionally, the present invention provides methods of producingrecombinant gelatins with varying degrees of cross-linking, useful forobtaining recombinant gelatins of desired melting points, gel strength,and viscosity.

[0154] The present invention provides methods to manipulate themolecular weight, the level of hydroxylation, and the degree ofcross-linking of the recombinant gelatins to allow for creation ofrecombinant gelatins of different and specific Bloom strengths, as wellas recombinant gelatins of different and specific levels of viscosity.

[0155] Proline hydroxylation plays central role in natural collagenformation. Hydroxylation of specific lysyl residues in the sequenceX-Lys-Gly also performs an important function in collagen synthesis andfibril formation. The hydroxyl groups on modified lysine residuesfunction as both attachment sites for carbohydrates and as essentialsites for the formation of stable intermolecular cross-links. Thesemodifications require the expression of specific enzymes, lysylhydroxylase and lysyl oxidase.

[0156] Therefore, in one aspect of the invention, the co-expression ofthese enzymes with the polypeptides of the present invention iscontemplated. The gene encoding lysyl hydroxylase (Hautala et al. (1992)Genomics 13:62-69) is expressed in a host cell, which is then furthermodified by the introduction of a sequence encoding a gelatin orpolypeptide from which gelatin can be derived, as described in thepresent invention. The recombinant gelatins of the present invention cantherefore be post-translationally modified by the activity ofendogenously expressed lysyl hydroxylase and lysyl oxidase. Therecombinant gelatins of the present invention can also be modified bythe expression of exogenous lysyl hydroxylase and lysyl oxidase. In oneembodiment, recombinant gelatins produced are non-hydroxylated, andsubsequently altered by imposing the desired degree of hydroxylation oflysine residues by the enzymatic activity of lysyl hydroxylase. Theability to alter the degree of lysyl hydroxylation is desirable inproducing gelatins, and polypeptides from which gelatin can be derived,with various degrees of cross-linking that lead to the desired gelstrengths and viscosities.

[0157] In further embodiments, a polypeptide containing hydroxylysineresidues can also be expressed in, for example, a yeast cell, in whichhydroxyproline is produced by the activity of prolyl hydroxylase. (SeeExamples 1 and 4.) In some embodiments, the modified recombinant gelatinor polypeptide from which gelatin can be derived can be formulated andadministered to an animal or human, thus serving as a substrate for theactivities of endogenous enzymes, such as lysyl oxidase, thus allowingthe collagenous polypeptide to be incorporated into tissues in astabilized cross-linked form. Therefore, one aspect of the presentinvention provides for the production of recombinant gelatins ofdesirable gel strengths and viscosity for commercial use, without theneed for lysyl hydroxylase or lysyl oxidase activities.

[0158] The invention also provides for the production of gelatin havinga particular gelling point. In one embodiment, the present methodsprovide for the production of gelatin having a setting or gelling pointof from 15 to 35° C. In further embodiments, the recombinant gelatin hasa setting point of from 15 to 25° C., from 25 to 35° C., and from 20 to30° C.

[0159] In various aspects, the present invention provides recombinantgelatin that is non-hydrolyzed, fully hydrolyzed, or hydrolyzed tovarying degrees, such as gelatins that are a mixture of hydrolyzed andnon-hydrolyzed products. Additionally, the present invention providesmethods of producing recombinant gelatins with varying degrees ofhydrolysis. (See Examples 9 and 10.) Gelatin hydrosylates are typicallycold water-soluble and are used in a variety of applications,particularly in the pharmaceutical and food industries, in which agelatin with non-gelling properties is desirable. Gelatin hydrolysatesare used in the pharmaceutical industry in film-forming agents,microencapsulation processes, arthritis and joint relief formulas,tabletting, and various nutritional formulas. In the cosmetics industry,gelatin hydrolysates are used in shampoos and conditioners, lotions andother formulations, including lipsticks, and in fingernail formulas,etc. Gelatin hydrolysates appear as nutritional supplements in proteinand energy drinks and foods; are used as fining agents in wine, beer,and juice clarification; and are used in the microencapsulation ofadditives such as food flavorings and colors. Gelatin hydrosylates areused in industrial applications for their film-forming characteristics,such as in coatings of elements in semiconductor manufacture, etc.

[0160] In one embodiment of the present invention, gelatin is producedfrom collagen sequences in which particular native domains have beendeleted or have been added in order to alter the behavior of theexpressed product. The invention further contemplates methods ofproducing recombinant gelatin wherein the gelatin is produced directlyfrom an altered collagen construct, without production of an intacttriple helical collagen. In particular, the present inventioncontemplates methods of producing recombinant gelatin comprising theexpression of various engineered constructs that do not encode standardtriple helical collagen. For example, specific deletions can eliminatecollagenase-responsive regions, and various regions elicitingimmunogenic, e.g., antigenic and allergenic, responses.

[0161] Specific domains of various collagens have been associated withspecific activities. (See, e.g., Shahan et al. (1999) Con. Tiss. Res.40:221-232; Raff et al. (2000) Human Genet 106:19-28, both of whichreferences are incorporated by reference herein in their entireties.) Inparticular, the present invention specifically provides for methods ofproducing recombinant gelatins derived from collagen constructs alteredto eliminate or to reduce or increase specific regions of a collagengene associated with a specific activity. Specifically, such regionscould be deleted in full or in part to produce a gelatin lacking or withreduced specific activity, or additional copies of the specific regioncould be added to produce a gelatin with enhanced activity. For example,sequences in types I and III collagen recognized by the α2β1 integrinreceptor on the platelet cell surface have been identified. (Knight etal. (1998) J. Biol. Chem. 273:33287-33294; and Morton et al. (1997) J.Biol. Chem. 272:11044-11048, which references are incorporated byreference herein in their entirety.)

[0162] In one aspect of the present invention, it is desirable to createa homogeneous gelatin composed of fragments synthesized from collagenconstructs lacking platelet activation regions. Such gelatin could beincluded, for example, in products associated with anastomosis andvascular grafting, etc., including coatings for stent and graft devices.Such products can be associated with deleterious side effects, forexample, thrombosis, that can develop in association with the use ofsuch products as a result of the platelet-aggregating regions present inthe collagenous product. In one aspect, the present invention providesfor a method of producing a recombinant gelatin which can providesupport for cell attachment when used in a stent or similar device, butwhich does not include platelet-reactive regions, thus minimizing therisk of platelet aggregation. (See Example 2.) Therefore, the presentinvention provides in one embodiment for a stent coating comprisingrecombinant gelatin. In a preferred embodiment, the recombinant gelatinis recombinant human gelatin. In some instances, such as various woundcare applications, it could be desirable to provide recombinant gelatincomprising domains capable of inducing specific aggregating activities.

[0163] A gelatin of the present invention could be expressed fromcollagen constructs that did not encode the regions recognized by theα2β1 receptor, or from constructs with one or with multiple copies ofsuch regions, thus providing a homogeneous and consistent gelatinproduct without or with reduced platelet aggregation and activation. Inone aspect, the present invention provides for the production ofrecombinant gelatin, either through direct expression of gelatin orthrough processing of gelatin from collagenous polypeptides, through theuse of highly efficient recombinant expression. The present productionmethods, as opposed to current methods of extraction, offer extremeflexibility, as any one of a number of expression systems can be used.The production material is accessible, for example, in yeast or plantbiomass. Secretion in certain production systems can be optimized, forexample, by dictating the uniform size of particular gelatin moleculesto be produced according to the present methods. In various embodiments,the present gelatins or the polypeptides from which these gelatins arederived, are produced in expression systems including, but not limitedto, prokaryotic expression systems, such as bacterial expressionsystems, and eukaryotic expression systems, including yeast, animal,plant, and insect expression systems. Expression systems such astransgenic animals and transgenic plants are contemplated.

[0164] The present invention provides for expression of at least onepolynucleotide encoding a gelatin or a polypeptide from which gelatincan be derived in a cell. In one embodiment, the present inventionprovides for the expression of more than one polynucleotide encoding agelatin or a polypeptide from which gelatin can be derived in a cell,such that recombinant gelatin containing homogeneous or heterogeneouspolypeptides is produced. The present invention further provides forexpression of a polynucleotide encoding a collagen processing orpost-translational enzyme or subunit thereof in a cell. Differentpost-translational modifications, and different post-translationalenzymes, e.g., prolyl hydroxylase, lysyl hydroxylase, etc., can effect,for example, Bloom strength and other physical characteristics of thepresent gelatins.

[0165] The recombinant gelatins of the present invention are derivedfrom collagenous sequences. The sequences from which the encodingpolynucleotides of the invention are derived can be selected from humanor from non-human sequences, depending on the characteristics desiredfor the intended use of the ultimate gelatin product. For pharmaceuticaland medical uses, recombinant human gelatin is preferred. Non-humansources include non-human mammalian sources, such as bovine, porcine,and equine sources, and other animal sources, such as chicken andpiscine sources. Non-native sequences are specifically contemplated.

[0166] Nucleic acid sequences encoding collagens have been generallydescribed in the art. (See, e.g., Fuller and Boedtker (1981)Biochemistry 20:996-1006; Sandell et al. (1984) J Biol Chem 259:7826-34;Kohno et al. (1984) J Biol Chem 259:13668-13673; French et al. (1985)Gene 39:311-312; Metsaranta et al. (1991) J Biol Chem 266:16862-16869;Metsaranta et al. (1991) Biochim Biophys Acta 1089:241-243; Wood et al.(1987) Gene 61:225-230; Glumoff et al. (1994) Biochim Biophys Acta1217:41-48; Shirai et al. (1998) Matrix Biology 17:85-88; Tromp et al.(1988) Biochem J 253:919-912; Kuivaniemi et al. (1988) Biochem J252:633-640; and Ala-Kokko et al. (1989) Biochem J 260:509-516.) (Seealso co-pending, commonly-owned application U.S. patent application Ser.No. 09/709,700, entitled “Animal Collagens and Gelatins,” filed 10 Nov.2000, incorporated herein by reference in its entirety.)

[0167] The nucleic acid sequences of the invention may be engineered inorder to alter the coding sequences used to produce recombinant gelatin,or polypeptides from which the recombinant gelatin can be derived, for avariety of ends including, but not limited to, alterations which modifyprocessing and expression of the gene product. For example, alternativesecretory signals may be substituted for any native secretory signals.Mutations may be introduced using techniques well known in the art,e.g., site-directed mutagenesis, PCR-directed mutagenesis, cassettemutagenesis, and other techniques well-known in the art to insert newrestriction sites, or to alter glycosylation patterns, phosphorylation,proteolytic turnover/breakdown, etc. Additionally, when producinggelatin in an expression system using particular host cells, thepolynucleotides of the invention may be modified in the silent positionof any triplet amino acid codon so as to better conform to the codonpreference of a particular host organism.

[0168] Altered polynucleotide sequences which may be used in accordancewith the invention include sequences containing deletions, additions, orsubstitutions of nucleotide residues in native collagen sequences. Suchpolynucleotides can encode the same or a functionally equivalent geneproduct. The gene product itself may contain deletions, additions orsubstitutions of amino acid residues within a collagen sequence.

[0169] The polynucleotide sequences of the invention are furtherdirected to sequences which encode variants of the encoded polypeptides.The encoded amino acid variants may be prepared by various methods knownin the art for introducing appropriate nucleotide changes for encodingvariant polypeptides. Two important variables in the construction ofamino acid sequence variants are the location of the mutation and thenature of the mutation. The amino acid sequence variants of the gelatinsof the present invention, or of the polypeptides from which the presentgelatins are derived, are preferably constructed by mutating thepolynucleotide to give an amino acid sequence that does not occur innature. These amino acid alterations can be made at sites that differin, for example, collagens from different species (variable positions),or in highly conserved regions (constant regions). Sites at suchlocations will typically be modified in series, e.g., by substitutingfirst with conservative choices (e.g., hydrophobic amino acid to adifferent hydrophobic amino acid) and then with more distant choices(e.g., hydrophobic amino acid to a charged amino acid), and thendeletions or insertions may be made at the target site.

[0170] Due to the inherent degeneracy of the genetic code, other nucleicacid sequences which encode substantially the same or a functionallyequivalent amino acid sequence or polypeptide, natural, synthetic,semi-synthetic, or recombinant in origin, may be used in the practice ofthe claimed invention. Degenerate variants are specifically contemplatedby the present invention, including codon-optimized sequences. Inaddition, the present invention specifically provides forpolynucleotides which are capable of hybridizing to a particularsequence under stringent conditions:

Expression

[0171] The present methods are suitably applied to the range ofexpression systems available to those of skill in the art. While anumber of these expression systems are described below, it is to beunderstood that application of the present methods not limited to thespecific embodiments set forth below.

[0172] A variety of expression systems may be utilized to contain andexpress sequences encoding the recombinant gelatins of the presentinventions or encoding polypeptides from which these gelatins can bederived. These include, but are not limited to, microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid, or cosmidnucleic acid expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors(e.g., baculovirus); filamentous fungi transformed with fungal vectors;plant cell systems transformed with virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or withbacterial expression vectors (e.g., pET or pBR322 plasmids); or animalcell systems.

[0173] Control elements or regulatory sequences suitable for use inexpressing the polynucleotides of the present invention are thosenon-translated regions of the vector, including enhancers, promoters,and 5′ and 3′ untranslated regions, which interact with host cellularproteins to carry out transcription and translation. Such elements mayvary in strength and specificity. Depending on the vector system andhost utilized, any number of suitable transcription and translationelements may be used.

[0174] Promoters are untranslated sequences located upstream from thestart codon of the structural gene that control the transcription of thenucleic acid under its control. Inducible promoters are promoters thatalter their level of transcription initiation in response to a change inculture conditions, e.g., the presence or absence of a nutrient. One ofskill in the art would know of a large number of promoters that would berecognized in host cells suitable for use in the methods of the presentinvention.

[0175] Promoter, enhancer, and other control elements can be selected assuitable by one skilled in the art. For example, when cloning inbacterial systems, inducible promoters such as the hybrid lacZ promoterof the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1plasmid (GIBCO BRL) and the like may be used. In insect cells, thebaculovirus polyhedrin promoter may be used. In plant systems, promotersor enhancers derived from the genomes of plant cells (e.g., heat shockpromoter, the promoter for the small subunit of RUBISCO; the promoterfor the chlorophyll a/b binding protein; promoters for various storageprotein genes, etc.) or from plant viruses (e.g., viral promoters orleader sequences, the 35S RNA promoter of CaMV, the coat proteinpromoter of TMV, etc.) may be cloned into the vector. In mammalian cellsystems, promoters from mammalian genes (e.g., metallothionein promoter,α-actin promoter, etc.) or from mammalian viruses (e.g., the adenoviruslate promoter, CMV, SV40, LTR, TK, and the vaccinia virus 7.5 Kpromoters, etc.) are preferable. If it is necessary to generate a cellline that contains multiple copies of the sequence encoding the desiredpolypeptide, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

[0176] Such promoters can be are operably linked to the polynucleotidesencoding the gelatin or gelatin precursors of the present invention,such as by removing the promoter from its native gene and placing theencoding polynucleotide at the 3′ end of the promoter sequence.Promoters useful in the present invention include, but are not limitedto, prokaryotic promoters, including, for example, the lactose promoter,arabinose promoter, alkaline phosphatase promoter, tryptophan promoter,and hybrid promoters such as the tac promoter; yeast promoters,including, for example, the promoter for 3-phosphoglycerate kinase,other glycolytic enzyme promoters (hexokinase, pyruvate decarboxylase,phophofructosekinase, glucose-6-phosphate isomerase, etc.), the promoterfor alcohol dehydrogenase, the alcohol oxidase (AOX) 1 or 2 promoters,the metallothionein promoter, the maltose promoter, and the galactosepromoter; and eukaryotic promoters, including, for example, promotersfrom the viruses polyoma, fowlpox, adenovirus, bovine papilloma virus,avian sarcoma virus, cytomegalovirus, retroviruses, SV40, and promotersfrom the target eukaryote, for example, the glucoamylase promoter fromAspergillus, actin or ubiquitin promoters, an immunoglobin promoter froma mammal, and native collagen promoters. (See, e.g., de Boer et al.(1983) Proc. Natl. Acad. Sci. USA 80:21-25; Hitzeman et al. (1980) J.Biol. Chem. 255:2073); Fiers et al. (1978) Nature 273:113; Mulligan andBerg (1980) Science 209:1422-1427; Pavlakis et al. (1981) Proc. Natl.Acad. Sci. USA 78:7398-7402; Greenway et al. (1982) Gene 18:355-360;Gray et al. (1982) Nature 295:503-508; Reyes et al. (1982) Nature297:598-601; Canaani and Berg (1982) Proc. Natl. Acad. Sci. USA79:5166-5170; Gorman et al. (1982) Proc. NatI. Acad. Sci. USA79:6777-6781; and Nunberg et al. (1984) Mol. and Cell. Biol.11(4):2306-2315.)

[0177] The polynucleotide sequences encoding the gelatins and gelatinprecursors of the present methods may be under the transcriptionalcontrol of a constitutive promoter, directing expression generally.Alternatively, the polynucleotides employed in the present methods areexpressed in a specific tissue or cell type, or under more preciseenvironmental conditions or developmental controls. Promoters directingexpression in these instances are known as inducible promoters. In thecase where a tissue-specific promoter is used, protein expression isparticularly high in the tissue from which extraction of the protein isdesired. In plants, for example, depending on the desired tissue,expression may be targeted to the endosperm, aleurone layer, embryo (orits parts as scutellum and cotyledons), pericarp, stem, leaves tubers,roots, etc. Examples of known tissue-specific promoters in plantsinclude the tuber-directed class I patatin promoter, the promotersassociated with potato tuber ADPGPP genes, the soybean promoter ofβ-conglycinin (7S protein), which drives seed-directed transcription,and seed-directed promoters from the zein genes of maize endosperm.(See, e.g., Bevan et al. (1986) Nucleic Acids Res. 14: 4625-4638; Mulleret al. (1990) Mol. Gen. Genet. 224: 136-146; Bray (1987) Planta172:364-370 ; and Pedersen et al. (1982) Cell 29:1015-1026.)

[0178] Transcription of the sequences encoding the gelatins or gelatinprecursors of the present invention from the promoter is often increasedby inserting an enhancer sequence in the vector. Enhancers arecis-acting elements, usually about from 10 to 300 bp, that act toincrease the rate of transcription initiation at a promoter. Manyenhancers are known for both eukaryotes and prokaryotes, and one ofordinary skill could select an appropriate enhancer for the host cell ofinterest. (See, e.g., Yaniv (1982) Nature 297:17-18.)

[0179] The gelatins and gelatin precursors of the present invention maybe expressed as secreted proteins. When the engineered cells used forexpression of the proteins are non-human host cells, it is oftenadvantageous to replace the secretory signal peptide of the collagenprotein with an alternative secretory signal peptide which is moreefficiently recognized by the host cell's secretory targeting machinery.The appropriate secretory signal sequence is particularly important inobtaining optimal fungal expression of mammalian genes. (See, e.g.,Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642.) Other signalsequences for prokaryotic, yeast, fungi, insect or mammalian cells arewell known in the art, and one of ordinary skill could easily select asignal sequence appropriate for the host cell of choice.

[0180] The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular cell system which is used, suchas those described in the literature. (See, e.g., Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162.) In addition, a host cellstrain may be chosen for its ability to modulate the expression of theinserted sequences or to process the expressed protein in the desiredfashion. Such modifications of the polypeptide include, but are notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation, prenylation, and acylation. Post-translational processingwhich cleaves a “prepro” form of the protein may also be used tofacilitate correct insertion, folding, and/or function. Different hostcells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specificcellular machinery and characteristic mechanisms for suchpost-translational activities, may be chosen to ensure the correctmodification and processing of the foreign protein.

[0181] In accordance with the invention, polynucleotide sequencesencoding recombinant gelatins or polypeptides from which gelatins can bederived may be expressed in appropriate host cells. In preferredembodiments of the invention, the recombinant gelatin is human gelatin.In other preferred embodiments of the invention, the polynucleotidesequences are derived from type I collagen sequence, free of codingsequence for any other type of collagen, or from type II collagen, freeof coding sequence for any other type of collagen, or from type IIIcollagen, free of coding sequence for any other type of collagen. Inanother embodiment, the encoding polynucleotides are derived from type Iand type III collagen in specified quantities, such that the gelatinproduced by or derived from the encoded polypeptides comprises a mixtureof type I and type III collagens in defined quantities.

[0182] In order to express the collagens from which the present gelatinsare derived, or to express sequences other than natural collagensequences leading to the production of the present gelatin, nucleotidesequences encoding the collagen, or a functional equivalent, or othersequence, for example, a shortened collagen sequence, such as thosepresented in Table 2, is inserted into an appropriate expression vector,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation.

[0183] Methods well-known to those skilled in the art can be used toconstruct expression vectors containing the desired coding sequence andappropriate transcriptional/translational control signals. These methodsinclude standard DNA cloning techniques, e.g., in vitro recombinanttechniques, synthetic techniques, and in vivo recombination. See, forexample, the techniques described in Maniatis et al., supra; Ausubel etal., supra; and Ausubel, F. M. (1997) Short Protocols in MolecularBiology, John Wiley and Sons, New York, N.Y.

[0184] Various expression vectors may be used to express the presentpolypeptides. For example, a typical expression vector contains elementscoding for a replication origin; a cloning site for insertion of anexogenous nucleotide sequence; elements that control initiation oftranscription of the exogenous gene, such as a promoter; and elementsthat control the processing of transcripts, such as atranscription/termination/polyadenylation sequence. An expression vectorfor use in the present invention can also contain such sequences as areneeded for the eventual integration of the vector into the chromosome.In addition, a gene that codes for a selection marker which isfunctionally linked to promoters that control transcription initiationmay also be within the expression vector, for example, an antibioticresistance gene to provide for the growth and selection of theexpression vector in the host.

[0185] The vectors of this invention may autonomously replicate in thehost cell, or may integrate into the host chromosome. Suitable vectorswith autonomously replicating sequences are well known for a variety ofbacteria, yeast, and various viral replications sequences for bothprokaryotes and eukaryotes. Vectors may integrate into the host cellgenome when they have a DNA sequence that is homologous to a sequencefound in host cell genomic DNA.

[0186] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the present polypeptides may be transformed using expressionvectors containing viral origins of replication or appropriateexpression elements (e.g., promoters, enhancers, transcriptionterminators, polyadenylation sites, etc.) and a selectable marker geneon the same or on a separate vector. Following the introduction of thevectors, cells may be allowed to grow for 1-2 days in enriched media,and are then switched to selective media. The selectable marker in therecombinant plasmid confers resistance to selection, allowing growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. This method mayadvantageously be used to produce cell lines which express a desiredpolypeptide.

[0187] Expression of the various sequences used in the methods of thepresent invention driven by, for example, the galactose promoters can beinduced by growing the culture on a non-repressing, non-inducing sugarso that very rapid induction follows addition of galactose; by growingthe culture in glucose medium and then removing the glucose bycentrifugation and washing the cells before resuspension in galactosemedium; and by growing the cells in medium containing both glucose andgalactose so that the glucose is preferentially metabolized beforegalactose-induction can occur.

[0188] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyl-transferase genes which can be employed in tk⁻ or aprt⁻cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell11:223-32; Lowy, I. et al. (1980) Cell 22:817-23.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. Therefore, the present invention contemplates the use of suchselectable markers, for example: dhfr, which confers resistance tomethotrexate; npt, which confers resistance to the aminoglycosidesneomycin and G-418; and als or pat, which confer resistance tochlorsulfuron and to phosphinotricin acetyltransferase, respectively.(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-3570; and Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14.)

[0189] Additional selectable genes have been described, for example,trpB, which allows cells to utilize indole in place of tryptophan, orhisD, which allows cells to utilize histinol in place of histidine.(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.Sci. 85:8047-51.) Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β-glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin, now widelyused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.)

[0190] As noted above, the expression vectors for use in the presentmethods of production can typically comprise a marker gene that confersa selectable phenotype on cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including atleast one set of genes coding for resistance to the antibioticspectinomycin, the streptomycin phophotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phophotransferase (NPTH) geneencoding kanamycin or geneticin resistance, the hygromycin resistancegene, genes coding for resistance to herbicides which act to inhibit theaction of acetolactate synthase (ALS), in particular, thesulfonylurea-type herbicides (e.g., the S4 and/or Hra mutations), genescoding for resistance to herbicides which act to inhibit action ofglutamine synthase, such as phophinothricin or basta (e.g. the bargene), or other similar genes known in the art. The bar gene encodesresistance to the herbicide basta, the nptII gene encodes resistance tothe antibiotics kanamycin and geneticin, and the ALS gene encodesresistance to the herbicide chlorsulfuron.

[0191] Other methods for determining which host cells, subsequent totransformation, contain the polynucleotides of interest include avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, nucleic acid hybridizations,including DNA-DNA or DNA-RNA hybridizations, and various proteinbioassay or immunoassay techniques including membrane-, solution-, orchip-based technologies for the detection and/or quantification ofpolynucleotides or polypeptides.

[0192] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellsthat possess the cellular machinery for proper processing of the primarytranscript, including various modifications such as protein folding,disulfide bond formation, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.

[0193] Specific initiation signals may also be used to achieve moreefficient translation of the polynucleotides of the present invention.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the present polypeptides, along with anyinitiation or upstream sequences required for translation, etc., areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequences, or portions thereof, are inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. (See, e.g., Bittner et al. (1987)Meth. in Enzymol. 153:516-544.)

[0194] A host cell of the present invention can be infected,transfected, or transformed with at least one polynucleotide encoding apost-translational enzyme, in addition to at least one polynucleotideencoding a gelatin of the present invention or a polypeptide from whichthe gelatin can be derived. Such polynucleotides include those encodingcollagen post-translational enzymes, such as prolyl 4-hydroxylase,collagen glycosyl transferase, C-proteinase, N-proteinase, lysyloxidase, or lysyl hydroxylase, and can be inserted into cells that donot naturally produce post-translational enzymes, for example, intoyeast cells, or cells that may not naturally produce sufficient amountsof post-translational enzymes, for example, various insect and mammaliancells, such that exogenous enzyme may be required to produce certainpost- translational effects. In one embodiment of the present invention,the post-translational enzyme is prolyl 4-hydroxylase, and thepolynucleotide encodes the α or the β subunit of prolyl hydroxylase. Ina preferred embodiment, polynucleotides encoding the α subunit and the62 subunit of prolyl 4-hydroxylase are inserted into a cell to produce abiologically active prolyl 4-hydroxylase enzyme, co-expressed with apolynucleotide encoding a gelatin or a polypeptide from which gelatincan be derived.

[0195] The polynucleotides encoding post-translational enzymes may bederived from any source, whether natural, synthetic, or recombinant. Ina preferred embodiment, the post-translational enzyme is derived fromthe same species as is the recombinant gelatin to be produced. In oneembodiment, the recombinant gelatin to be produced is human recombinantgelatin, and the post translational enzyme is human prolyl4-hydroxylase.

[0196] The expressed gelatins or gelatin precursors of the presentinvention are preferably secreted into culture media and can be purifiedto homogeneity by methods known in the art, for example, bychromatography. In one embodiment, the recombinant gelatin or gelatinprecursors are purified by size exclusion chromatography. However, otherpurification techniques known in the art can also be used, including,but not limited to, ion exchange chromatography, hydrophobic interactionchromatography (HIC), and reverse-phase chromatography. (See, e.g.,Maniatis et al., supra; Ausubel et al., supra; and Scopes (1994) ProteinPurification: Principles and Practice, Springer-Verlag New York, Inc.,NY.)

Prokaryotic

[0197] In prokaryotic systems, such as bacterial systems, any one of anumber of expression vectors may be selected, depending upon the useintended for the polypeptides to be expressed. For example, when largequantities of the recombinant gelatins of the present invention, orpolypeptides from which these recombinant gelatins can be derived, areneeded, vectors which direct high-level expression of fusion proteinsthat can be readily purified may be used. Such vectors include, but arenot limited to, multifunctional E. coli cloning and expression vectorssuch as BLUESCRIPT (Stratagene), in which the encoding sequence may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent seven residues of β-galactosidase so that ahybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX(Promega, Madison, Wis.) and pET (Invitrogen) vectors may also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by a variety of methods known inthe art, for example, by adsorption to glutathione-agarose beadsfollowed by elution in the presence of free glutathione. Proteins madein such systems may be designed to include heparin, thrombin, or factorXA protease cleavage sites so that the cloned polypeptide of interestcan be released from the GST moiety.

Yeast

[0198] In preferred embodiments, the present invention provides methodsof producing recombinant gelatin using a yeast expression system. Inpreferred embodiments, gelatin is produced directly from alteredcollagen constructs or derived from processing of collagenouspolypeptides. A number of vectors containing constitutive,non-consitutive, or inducible promoters may be used in yeast systems.(See, e.g., Ausubel et al., supra, Chapter 13.) In some aspects, vectorscontaining sequences which direct DNA integration into the chromosomeare used for expression in S. cerevisiea.

[0199] In one embodiment, the recombinant gelatins of the invention, orthe polypeptides from which these gelatins can be derived, are expressedusing host cells from the yeast Saccharomyces cerevisiae. Saccharomycescerevisiae can be used with any of a large number of expression vectorsavailable in the art, including a number of vectors containingconstitutive or inducible promoters such as α factor, AOX, GAL1-10, andPGH. (See, e.g., Ausubel et al., supra, and Grant et al. (1987) MethodsEnzymol. 153:516-544.) Commonly employed expression vectors are shuttlevectors containing the 2μ origin of replication for propagation both inyeast and the ColEl origin for E. coli, including a yeast promoter andterminator for efficient transcription of the foreign gene. Vectorsincorporating 2μ plasmids include, but are not limited to, pWYG4 andpYES2, which have the 2μ ORI-STB elements, the GAL1-10, etc. In onemethod of the present invention, in which a hydroxylated product isdesired, involves the co-expression of a collagen post-translationalenzyme, for example, prolyl 4-hydroxylase. In one such method, using thepWYG4 vector, the Ncol cloning site is used to insert the gene foreither the α or β subunit of prolyl 4-hydroxylase, and to provide theATG start codon for either the α or β subunit. In one method, expressionplasmids are used which direct integration into the chromosome of thehost.

[0200] The expression vector pWYG7L, which has intact 2α ORI, STB, REP1and REP2, the GAL7 promoter, and the FLP terminator, can also be used.When the co-expression of a post-translational enzyme, for example,prolyl 4-hydroxylase, is desired, the gene for either the α or α subunitof prolyl 4-hydroxylase is inserted in the polylinker with its 5′ endsat a BamHI or Ncol site. The vector containing the prolyl 4-hydroxylasegene is transformed into S. cerevisiae either before or after removal ofthe cell wall to produce spheroplasts that take up DNA on treatment withcalcium and polyethylene glycol or by treatment of intact cells withlithium ions. Alternatively, DNA can be introduced by electroporation.Transformants can be selected by using host yeast cells that areauxotrophic for leucine, tryptophane, uracil or histidine together withselectable marker genes such as LEU2, TRP1, URA3, HIS3 or LEU2-D.

[0201] In another preferred embodiment, the methods of producingrecombinant gelatin according to the present invention use host cellsfrom the yeast Pichia pastoris, or from other species ofnon-Saccharomyces yeast, that possess advantages in producing highyields of recombinant protein in scaled-up procedures. Pichia expressionsystems include advantages of both prokaryotic (e.g., E. coli)expression systems—high-level expression, easy scale-up, and inexpensivegrowth—and eukaryotic expression systems—protein processing, folding,and post-translational modifications. Such expression systems can beconstructed using various methods and kits available to those skilled inthe art, for example, the PICHIA EXPRESSION kits available fromInvitrogen Corporation (San Diego, Calif.).

[0202] There are a number of methanol responsive genes in methylotrophicyeasts such as Pichia pastoris, or Pichia methanolica, etc., theexpression of each being controlled by methanol responsive regulatoryregions (also referred to as promoters). Any of such methanol responsivepromoters are suitable for use in the practice of the present invention.Examples of specific regulatory regions include the promoter for theprimary alcohol oxidase gene from Pichia pastoris AOX1, the promoter forthe secondary alcohol oxidase gene from Pichia pastoris AOX2, the FLD 1promoter, the promoter for the dihydroxyacetone synthase gene fromPichia pastoris (DAS), the promoter for the P40 gene, etc. Typically,expression in Pichia pastoris is obtained by the promoter from thetightly regulated AOX1 gene. (See, e.g., Ellis et al. (1985) Mol. Cell.Biol. 5: 1111; and U.S. Pat. No. 4,855,231.) Constitutive expression canalso be achieved using, e.g., the GPH promoter.

[0203] Another yeast expression system preferred for use in the methodsof the present invention makes use of the methylotrophic yeast Hansenulapolymorpha. This system can be used, for example, in a method ofproduction of the present invention where high yield is desirable.Growth on methanol results in the induction of enzymes key in, such asMOX (methanol oxidase), DAS (dihydroxyacetone synthase), and FMHD(formate dehydrogenase). These enzymes can constitute up to 30-40% ofthe total cell protein. The genes encoding MOX, DAS, and FMDH productionare controlled by strong promoters induced by growth on methanol andrepressed by growth on glucose. Any or all three of these promoters maybe used to obtain high level expression of heterologous sequences in H.polymorpha, according to methods known in the art.

[0204] In one method of the present invention, the encodingpolynucleotides are cloned into an expression vector under the controlof an inducible H. polymorpha promoter. If secretion of the product isdesired, a polynucleotide encoding a signal sequence for secretion inyeast, such as MFα1, is fused in frame with the coding sequence for thepolypeptides of the invention. The expression vector preferably containsan auxotrophic marker gene, such as URA3 or LEU2, or any other markerknown in the art, which may be used to complement the deficiency of anauxotrophic host. Alternatively, dominant selectable markers such aszeocin or blastacin may be used.

[0205] The expression vector is then used to transform H. polymorphahost cells using techniques known to those of skill in the art. Aninteresting and useful feature of H. polymorpha transformation is thespontaneous integration of up to 100 copies of the expression vectorinto the genome. In most cases, the integrated sequences form multimersexhibiting a head-to-tail arrangement. The integrated foreign DNA hasbeen shown to be mitotically stable in several recombinant strains, evenunder non-selective conditions. This phenomenon of high copy integrationfurther adds to the productivity potential of the system.

Plant

[0206] The present invention also contemplates the production of therecombinant gelatin of the present invention, or polypeptides from whichthe recombinant gelatin can be derived, in plant expression systems,including plant host cells and transgenic plants. (See, e.g., TransgenicPlants: A Production System for Industrial and Pharmaceutical Proteins,Owen and Pen, eds., John Wiley & Sons, 1996; Transgenic Plants, Galunand Breiman, eds., Imperial College Press, 1997; and Applied PlantBiotechnology, Chopra et al. eds., Science Publishers, Inc., 1999.) Incases where plant expression vectors are used, the expression ofsequences may be driven by any of a number of promoters. For example,viral promoters such as the 35S and 19S promoters of CaMV may be usedalone or in combination with the omega leader sequence from TMV. (See,e.g., Brisson et al. (1984) Nature 310:511-514; and Takamatsu, N. (1987)EMBO J. 6:307-311.) Plant expression vectors and reporter genes aregenerally known in the art. (See, e.g., Gruber et al. (1993) in Methodsof Plant Molecular Biology and Biotechnology, CRC Press.)

[0207] Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters e.g., soybean hsp17.5-E or hsp17.3-B maybe used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991)Results Probl. Cell Differ. 17:85-105; and Gurley et al. (1986) Mol.Cell. Biol. 6:559-565.) These constructs can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, pathogen-mediatedtransfection, particle bombardment, or any other means known in the art,such as are described in a number of generally available reviews. (See,e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y., pp. 191-196; Weissbachand Weissbach (1988) Methods for Plant Molecular Biology, AcademicPress, NY, Section VIII, pp. 421-463; and Grierson and Corey, PlantMolecular Biology, 2d Ed., Blackie, London, Ch. 7-9.)

[0208] In various embodiments, the recombinant gelatin of the presentinvention, or polypeptides from which the present recombinant gelatincan be derived, is produced from seed by way of available seed-basedproduction techniques using, for example, canola, corn, soybeans, rice,and barley seed. In such embodiments, the protein is recovered duringseed germination/molting. In other embodiments, the protein is expresseddirectly into the endosperm or into other parts of the plant so that thegelatin is non-extracted, and the plant itself can serve as, forexample, a dietary supplement such as a source of protein.

[0209] Promoters that may be used to direct the expression of thepolynucleotides may be heterologous or non-heterologous. These promoterscan also be used to drive expression of antisense nucleic acids toreduce, increase, or alter expression as desired. Other modificationsmay be made to increase and/or maximize transcription of sequences in aplant or plant cell are standard and known to those in the art. Forexample, the polynucleotide sequences operably linked to a promoter mayfurther comprise at least one factor that modifies the transcriptionrate of the encoded polypeptides, such as, for example, peptide exportsignal sequence, codon usage, introns, polyadenylation signals, andtranscription termination sites. Methods of modifying nucleic acidconstructs to increase expression levels in plants are generally knownin the art. (See, e.g. Rogers et al. (1985) J. Biol. Chem. 260:3731;Cornejo et al. (1993) Plant Mol Biol 23:567-568.) In engineering a plantsystem that affects the rate of transcription of the polynucleotides,various factors known in the art, including regulatory sequences such aspositively or negatively acting sequences, enhancers and silencers,chromatin structure, etc., can be used.

[0210] Typical vectors useful for expression of foreign genes in plantsare well known in the art, including, but not limited to, vectorsderived from the tumor-inducing (Ti) plasmid of Agrobacteriumtumefaciens. These vectors are plant integrating vectors, that upontransformation, integrate a portion of the DNA into the genome of thehost plant. (See, e.g., Rogers et al. (1987) Meth. In Enzymol.153:253-277; Schardl et al. (1987) Gene 61:1-11; and Berger et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:8402-8406.)

[0211] Procedures for transforming plant cells are available in the art,including, for example, direct gene transfer, in vitro protoplasttransformation, plant virus-mediated transformation, liposome-mediatedtransformation, microinjection, electroporation, Agrobacterium-mediatedtransformation, and ballistic particle acceleration. (See, e.g.,Paszkowski et al. (1984) EMBO J. 3:2717-2722; U.S. Pat. No. 4,684,611;European Application No. 0 67 553; U.S. Pat. No. 4,407,956; U.S. Pat.No. 4,536,475; Crossway et al. (1986) Biotechniques 4:320-334; Riggs etal. (1986) Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee et al. (1988)Biotechnology 6:915-921; and U.S. Pat. No. 4,945,050.) Standard methodsfor the transformation of rice, wheat, corn, sorghum, and barley aredescribed in the art. (See, e.g., Christou et al. (1992) Trends inBiotechnology 10:239; Casas et al. (1993) Proc. Nat'l Acad. Sci. USA90:11212; Wan et al. (1994) Plant Physiol. 104:37; and Lee et al. (1991)Proc. Nat'l Acad. Sci. USA 88: 6389.) Wheat can be transformed bytechniques similar to those employed for transforming corn or rice.(See, e.g., Fromm et al. (1990) Bio/Technology 8:833; and Gordon-Kamm etal., supra.)

[0212] Additional methods that may be used to generate plants or plantcells that can express the present recombinant gelatins, or polypeptidesfrom which these recombinant gelatins can be derived, arewell-established in the art. (See, e.g., U.S. Pat. No. 5,959,091; U.S.Pat. No. 5,859,347; U.S. Pat. No. 5,763,241; U.S. Pat. No. 5,659,122;U.S. Pat. No. 5,593,874; U.S. Pat. No. 5,495,071; U.S. Pat. No.5,424,412; U.S. Pat. No. 5,362,865; and U.S. Pat. No. 5,229,112.)

[0213] The present invention further provides a method of producingpolypeptides by providing a biomass from plants or plant cells which arecomprised of at least one polynucleotide sequence encoding a recombinantgelatin, or a polypeptide from which recombinant gelatin can be derived,wherein such polynucleotide sequence is operably linked to a promoter toeffect the expression of the polypeptide. In a further embodiment, themethod additionally comprises co-expression of at least onepolynucleotide sequence encoding an enzyme that catalyzes apost-translational modification, or subunit thereof, wherein suchpolynucleotide sequence is operably linked to a promoter. In thesemethods, the recombinant gelatins or collagenous polypeptides areextracted from the biomass.

Fungi

[0214] Filamentous fungi may also be used to produce the polypeptides ofthe instant invention. Vectors for expressing and/or secretingrecombinant proteins in filamentous fungi are well known in the art, andone of skill in the art could, using methods and products available inthe art, use these vectors in the presently recited methods. (See, e.g.,U.S. Pat. No. 5,834,191.)

Insect

[0215] Insect cell systems allow for the polypeptides of the presentinvention to be produced in large quantities. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes in, for example, Spodoptera frugiperdacells or in Trichoplusia larvae. Sequences encoding the gelatins orgelatin precursors of the present invention may be cloned intonon-essential regions of the virus, for example, the polyhedron gene,and placed under control of an AcNPV promoter, for example, thepolyhedron promoter. Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat encoded by the polyhedron gene). These recombinant viruses are thenused to infect Spodoptera frugiperda cells or Trichoplusia larvae inwhich polynucleotides encoding the gelatins or gelatin precursors areexpressed. (See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat. Acad.Sci. 91:3224-3227; Smith et al. (1983) J. Virol. 46:584; and U.S. Pat.No.4,215,051). Further examples of this expression system may be foundin, e.g. Ausubel et al. (1995), supra.

[0216] Recombinant production of the polypeptides of the presentinvention can be achieved in insect cells, for example, by infection ofbaculovirus vectors containing the appropriate polynucleotide sequences,including those encoding any post-translational enzymes that might benecessary. Baculoviruses are very efficient expression vectors for thelarge-scale production of various recombinant proteins in insect cells.Various methods known in the art can be employed to construct expressionvectors containing a sequence encoding a gelatin or gelatin precursor ofthe present invention and the appropriate transcriptional/translationalcontrol signals. (See, e.g., Luckow et al. (1989) Virology 170:31-39;and Gruenwald, S. and J. Heitz (1993) Baculovirus Expression VectorSystem: Procedures & Methods Manual, Pharmingen, San Diego, Calif.)

Animal

[0217] The present invention provides methods of expressing therecombinant gelatins of the present invention, or polypeptides fromwhich the recombinant gelatins of the present invention can be derived,in animal systems. Such systems include mammalian and non-vertebratehost cells and transgenic animals. In mammalian host cells, a number ofexpression systems may be utilized. In cases where an adenovirus is usedas an expression vector, sequences encoding the polypeptides of thepresent invention may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential E1 or E3 region of the viral genome may be used toobtain a viable virus which is capable of expressing the polypeptides ofthe present invention in infected host cells. (See, e.g., Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) Alternatively,the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et al.(1982) Proc. Natl. Acad. Sci. USA 79:7415-7419 (1982); Mackett et al.(1984), J. Virol. 49:857-864; and Panicali et al., (1982) Proc. Natl.Acad. Sci. USA 79:4927-4931.) In addition, various transcriptionenhancers known in the art, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in, for example, mammalianhost cells.

[0218] Semliki Forest virus is a preferred expression system as thevirus has a broad host range such that infection of mammalian cell lineswill be possible. Infection of mammalian host cells, for example, babyhamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells, usingsuch a viral vector can yield very high recombinant expression levels.More specifically, it is contemplated that Semliki Forest virus can beused in a wide range of hosts, as the system is not based on chromosomalintegration, and therefore will be a quick way of obtainingmodifications of the recombinant gelatins in studies aimed atidentifying structure-function relationships and testing the effects ofvarious hybrid molecules. Methods for constructing Semliki Forest virusvectors for expression of exogenous proteins in mammalian host cells areknown in the art and are described in, for example, Olkkonen et al.(1994) Methods Cell Biol 43:43-53.

[0219] Additionally, CHO cells deficient in dihydrofolate reductase(dhfr) can be transfected with an expression plasmid containing a dhfrgene and the desired polynucleotide. Selection of CHO cells resistant toincreasing concentrations of methotrexate will undergo geneamplification, providing higher expression levels of the desiredrecombinant protein, as known in the art.

[0220] Transgenic animal systems may also be used to express therecombinant gelatins of the present invention or the polypeptides fromwhich these recombinant gelatins can be derived. Such systems can beconstructed, for example, in mammals by operably linking an encodingpolypeptide to a promoter and other required or optional regulatorysequences capable of effecting expression in mammary glands. Likewise,required or optional post-translational enzymes that effectpost-translational modifications, may be produced simultaneously in thetarget cells employing suitable expression systems. Methods of usingtransgenic animals to recombinantly produce proteins are known in theart. (See, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,824,838; U.S.Pat. No. 5,487,992; and U.S. Pat. No. 5,614,396; and co-pending U.S.patent application Ser. No. 08/987,292.)

Vaccine Formulations

[0221] The present invention specifically contemplates the use of therecombinant gelatins of the present invention as components of variouspharmaceutical products, including vaccines. (See, e.g., co-pending,commonly-owned application U.S. patent application Ser. No. 09/710,239,entitled “Recombinant Gelatins,” filed 10 Nov. 2000, incorporated byreference herein in its entirety.) Therefore, in one aspect, the presentinvention provides a vaccine formulation comprising recombinant gelatin.In a preferred embodiment, the recombinant gelatin is derived from humansources. The present recombinant gelatins offer an advantage previouslyunavailable in the art: that of using gelatins derived from native humancollagen sequence, thus reducing the risk of immunogenecity and/orantigenicity from the gelatin itself. In a specific embodiment of thepresent invention, a vaccine comprising a non-immunogenic recombinantgelatin is provided. (See Example 12.) In a further embodiment, thenon-immunogenic recombinant gelatin is derived from human sequence. Inother embodiments, the non-immunogenic recombinant gelatin is derivedfrom animal sources, e.g., from bovine or porcine sequences. In furtherembodiments, the non- immunogenic recombinant gelatin comprises anon-native sequence.

[0222] In addition, as the present gelatins are produced recombinantlyin a controlled environment, the risk of infectivity, from infectiveagents such as TSEs, pathologic bacteria, or viruses, or the risk ofexposure to endotoxins and pathogens introduced during or left over fromprocessing, are virtually eliminated. It is another object of theinvention to provide improved stabilizing agents which includerecombinant gelatins, and derivatives, fragments, or functionalequivalents thereof, each with physical and chemical characteristicsoptimized for particular applications.

[0223] Stability is a critical issue in vaccine development, where therelative instability of most existing viruses must be addressed.Vaccines can degrade over time, losing potency and effectiveness. Inaddition, vaccines are distributed worldwide, and in a diverse range ofenvironments and conditions. Delivery of vaccines in developingcountries, for example, has sometime been problematic due to imperfectstorage conditions, including, e.g., high ambient temperatures. Undercertain storage and/or transport conditions, the stability of a vaccineformulation can be compromised. (See, e.g., Chang, A. C. et al. (1996)J. Pharm. Sci. 85:129-132; Koyama, K. et al. (1996); Osame, J., EuropeanPatent No. 0 568 726 B1; and U.S. Pat. No. 4,337,242.)

[0224] Vaccine stabilization refers to the maintenance of the safety andeffectiveness of a vaccine. Stabilizing agents are thus compounds thatperform a variety of different tasks. For example, with live vaccines,such as those for measles, mumps, rubella, and canine distemper,stability involves maintenance of the infectious titer/infectivity ofthe vaccine and the ability to elicit the appropriate immune response.With inactivated vaccines, such as those for influenza, Hepatitis A,poliomyelitis (IPV), and rabies, as well as in vaccines produced frompurified bacterial protein toxins, such as against cholera andpertussis, the ability to induce the desired immune response requiresmaintenance of the structural integrity of the vaccine, in order topreserve antigenic structure and correct steric presentation of relevantepitopes. Therefore, there is a need for effective stabilization ofvaccine preparations in anticipation of varying lengths of storage, andvarying conditions of storage, transport, and use.

[0225] Current stabilization methods seek to improve storage and heatstability, boost immunological responses to the infectious agent byensuring proper in vivo delivery, and provide formulations having thepotential for reducing the number of immunizations required forprotective efficacy. Several stabilization methods, such as the use oflow temperatures, lyophilization processes, and chemical stabilizers,have been used in the past. Low temperature storage facilities, forexample, offering storage conditions of from about −10° C. to −70° C.,can provide one method of maintaining the stability of vaccineformulations. However, such facilities are not always available duringthe transport or distribution of vaccines. Maintaining a proper “coldchain” of storage facilities can be difficult in view of the properinfrastructure required to monitor and maintain the necessary equipment,and to protect the vaccine formulation from development through toadministration.

[0226] Another stabilization technique involves the use of variouslyophilization or freeze-drying procedures. Lyophilization is areasonably effective but expensive procedure. The lyophilization processrequires three distinct stages (freezing, primary drying, and secondarydrying), each of which presents challenges for maintaining the nativeconformation and activity of biological macromolecules andmicroorganisms. (See, e.g., Burke et al. (1999) Crit. Rev. Ther. DrugCarrier Syst. 16:1-83, and references therein.) Lyophilized vaccinesremain fairly stable at about 4° to 8° C., but undergo deteriorationthroughout the storage period. Lyophilized vaccines slowly deteriorateuntil, at around 12 to 24 months of storage, the vaccine formulationlacks sufficient titer to confer immunization. In addition, lyophilizedvaccine formulations must be reconstituted prior to use. Furthermore,the liquid reconstituted preparation loses potency while at roomtemperature. This can result in an imperfect immune response, and canlead to an increase in the number of immunizations required forprotective efficacy.

[0227] In another approach to confer stability, stabilizing agents areadded to a vaccine formulation and used in conjunction with either lowertemperature storage or lyophilization methods. These stabilizers canperform a variety of functions directed to maintenance of stability,including, for example, maintaining formulation pH, contributing toviscosity and/or tonicity, assisting in controlled release, minimizingaggregation and precipitation of formulation components, and hydratingformulation components, to prevent damage caused by dehydration duringdesiccation and/or subsequent long-term storage, which can includepreferential hydration of protein constituents to maintain nativeprotein conformation. In order to maximize the stabilizing effects ofthese materials, stabilizers are often employed in combination, so thatthe stabilizing effect can be synergistically or additively increased.

[0228] Stabilizers used in the formulation of vaccines thus include, butare not limited to, buffers; salts, such as sodium chloride or magnesiumchloride; amino acids, such as sodium glutamate, arginine, lysine, andcysteine; monosaccharides, such as glucose, galactose, fructose, andmannose; disaccharides, such as sucrose, maltose, and lactose; sugaralcohols, such as sorbitol, and mannitol; polysaccharides, such asoligosacchandes, starch, cellulose, and derivatives thereof; human serumalbumin and bovine serum albumin; various antioxidants, such as ascorbicacid; and gelatin, such as hydrolyzed gelatin.

[0229] Hydrolyzed gelatin has been employed as a component of vaccines,for example, as a stabilizing agent for liquid and lyophilized vaccineformulations. (See, e.g., U.S. Pat. No. 4,985,244; U.S. Pat. No.4,147,772; U.S. Pat. No. 4,338,335; and Makino, S. et al. EuropeanPatent No. 0 295 043 B 1.) Partially hydrolyzed gelatins currentlyavailable are derived from gelatins produced by conventional extractionmethods from raw animal sources. (See, e.g., Asghar, A. (1982) Adv. FoodRes. 28:231-372; and Miller, E. J. et al. (1982) Methods in Enzymology82:33-64.)

[0230] Gelatin can serve to stabilize vaccines, for example, lyophilizedvaccines, at different stages. Lyophilization involves various extremes,including extremes of temperature, pH, salt concentration, etc. Redoxreactions can occur as the solvent is removed, and the solutes areconcentrated and dried. Various characteristics of gelatin, includingits surface-active, gluing, emulsifying, and viscosity-enhancingproperties, can contribute to its stabilizing effects when used in suchcircumstances.

[0231] While lyophilized animal-source gelatins have been used with somesuccess, these are essentially proteins foreign to the human system, andtherefore capable of causing undesirable antigenic and/or immunogenicresponses. Moreover, currently available materials are plagued byproblems of variability and purity. Concern over the presence ofinfective agents such as TSEs in animal-derived proteins has led togovernmental and regulatory restrictions relating to the recovery anduse of such materials. For example, endotoxin levels of commercialmaterials typically range from about 1.0 to 1.5 EU/mg of gelatin. (See,e.g., Schaegger, H. and G. von Jagow (1987) Anal. Biochem. 166:368-379;and Friberger, P. et al. (1987) in “Detection of Bacterial Endotoxinswith the Limulus Ameobocyte Lysate Test,” Prog. Clin. Biol. Res.231:149-169.)

[0232] The present invention provides recombinant gelatins that can beused as a component of vaccines, and methods for producing suchrecombinant gelatins. In one aspect, the recombinant gelatin is humanrecombinant gelatin. In the methods of the present invention, theendotoxin levels can be reduced by two to three orders of magnitude fromthose of commercially available gelatins derived from animal sources.(See Example 8.) The present invention thus provides, in one embodiment,a recombinant gelatin that is virtually endotoxin-free.

[0233] Furthermore, the present invention specifically provides anon-immunogenic gelatin. In one embodiment, the present inventionprovides for vaccine comprising recombinant gelatin, wherein therecombinant gelatin is human gelatin. Use of such a material as acomponent in vaccine formulations can reduce the risk of immunogenecitycurrently presented by the use of animal-derived gelatin components.(See, e.g., Sakaguchi and Inouye, supra; Sakaguchi et al., supra;Nakayama et al., supra; Asher, supra; and Verdrager, supra.) The use ofgelatins derived from native human sequences will reduce the likelihoodof a post-administration immunogenic response to the gelatin product. Inone embodiment, the present invention provides for the use ofrecombinant gelatins encoded by altered collagen constructs, wherein theencoded gelatins are nonimmunogenic. (See Example 12) Such recombinantgelatins can be constructed using assays designed to identify regionsresponsible for inducing immonogenic and antigenic responses, and therecombinant gelatins of the present invention can be specificallytailored to avoid these domains.

[0234] In addition to providing a gelatin material without theantigenicity, immunogenecity, and infectivity risks associated with theuse of animal-derived materials, the present invention allows for areproducible source of consistent product. Specifically, the presentgelatins can be presented as a homogeneous mixture of identicalmolecules. The physical characteristics desired in a particular medicalapplication can be specifically introduced and achieved consistently.The present invention is thus able to provide a reliable and consistentproduct will minimize variability associated with the availability anduse of current gelatin products.

[0235] The recombinant gelatins of the present invention can be designedto possess specific physical properties suitable for use in particularapplications. The present invention provides methods for varyingcharacteristics such as molecular weight, gel strength, and pH of thefinal gelatin formulation to produce gelatins with specific propertiesas desired, and to thus meet customer's specifications to a degreeunattainable with currently available materials. Moreover, suchformulations allow the customer to explore refinements of existingprocesses and formulations, as well as to develop new applications, forthe present recombinant gelatins.

[0236] With respect to the problem of purity, the present inventionprovides recombinant gelatins that are virtually endotoxin-free.(Example 8.) Furthermore, risks associated with variability andassociated problems in reproducibility and predictability of behavior,are virtually eliminated as the recombinant gelatins of the presentinvention can comprise gelatin polypeptides with well-definedcharacteristics and behaviors. In one embodiment, the present inventionprovides a composition comprising a homogeneous mixture of recombinantgelatin polypeptides. In a further embodiment, the present inventionprovides a heterogeneous mixture of recombinant gelatin polypeptides.

[0237] In one embodiment, the invention encompasses recombinant gelatinswith specific molecular weights. In another embodiment, the presentinvention provides for recombinant gelatins possessing particular rangesof molecular weights. (See Example 1.) For example, the presentinvention allows for the production of recombinant human solublegelatins with molecular weight distributions similar to those ofcurrently available animal-derived soluble gelatins used in theformulation of vaccines as stabilizers. (See Examples 1, 7, and 9.)

[0238] Recombinant gelatins comprising uniform polypeptides of specificmolecular weights are specifically provided. Gelatins currently used invaccines are typically hydrolyzed fragments of approximately 60 kDa andless. Such fragments are preferred, for example, to reduce the risk ofimmunogenic response to the gelatin. The present gelatins can beprovided as small molecular weight polypeptides. In addition, using themethods of the present invention, specific non-immunogenic recombinantgelatins can be constructed, by identifying regions in a collagen orcollagenous sequence responsible for inducing an immunogenic response,and producing a recombinant gelatin that does not contain that region.(See, e.g., Example 12.)

[0239] It is to be noted that gelatin is used in the processing andpreparation of vaccines, as well as appearing as a component of vaccineformulations. The recombinant gelatins of the present invention can beused in preparation and processing steps, and provide distinctadvantages over currently used animal-derived products. For example, thegelatins of the present invention can be produced as consistentmolecules with defined and characterized properties, limitingvariability in performance and allowing for fine-tuning andreproducibility of manufacturing procedures.

[0240] The molecular weight distributions of commercially availableanimal-derived soluble gelatins, such as those used in formulation ofvaccines, range from about 0 to 30 kDa and from about 0 to 60 kDa. (SeeExample 9.) The present invention provides a method of producingrecombinant human gelatins with molecular weight distributions thatcorrespond to those of commercially available gelatins, and can be usedfor the same purposes. Additionally, the present invention providesmethods for producing gelatins with a narrower molecular weightdistribution, for example, about 10 to 30 kDa, or about 30 to 50 kDa,not available from commercial materials.

[0241] The present invention provides multiple methods of producing therecombinant gelatin of the appropriate molecular weight for use in acertain application. For example, recombinant gelatin can be derivedthrough processing of recombinant collagen, such as by hydrolysistechniques, including acid, thermal, or enzymatic hydrolysis. (SeeExamples 9 and 10.) In another aspect, the recombinant gelatin isnon-hydrolyzed, and the recombinant gelatin of the appropriate size isobtained through expression of recombinant gelatin from an alteredcollagen construct. (See Example 1.)

[0242] The present invention provides for the production of recombinantgelatins specifically tailored, through manipulation of cross-linking,hydroxylation, molecular weight, or any combination thereof, to possessdefined thermal characteristics suited for particular applications. Inone aspect, the invention provides recombinant gelatin, suitable for useas a stabilizing agent or excipient for vaccines, that improves thermalstability, even when these vaccines are subjected to high temperaturesfor long periods. In another aspect, the present invention providesstabilizing agents suitable for use in association with certain vaccineswhose active components are thermo-sensitive, including viral vaccinessuch as, for example, yellow fever. In one embodiment, the presentinvention provides a recombinant gelatin that stabilizes vaccines atabove sub-zero temperatures.

[0243] It is to be noted that the present invention specificallycontemplates that the present recombinant gelatins can provide anacceptable source of material for use by populations whose religious orother dictates preclude the use of materials derived from particularanimal sources, e.g., from bovine or from porcine sources.

[0244] In one embodiment, the vaccines of the present invention can bemanufactured in ready-to-use form, such as in filled syringes or inmicroencapsulated form, eliminating the need for subsequent processingprior to use.

[0245] Hydrolyzed gelatin is used as a stabilizer in current vaccineformulations. The present invention provides in one embodimentrecombinant gelatin with characteristics similar to those of hydrolyzedanimal-source gelatin, e.g., similar molecular weights, meltingtemperatures, etc. These recombinant gelatins can be produced throughhydrolysis of recombinant collagen, or can be produced directly fromaltered collagen constructs. The present invention provides gelatinsthat are reproducible and well-characterized proteins with definedproperties. Therefore, the recombinant gelatins used, in the preparationand manufacture of vaccines and/or as stabilizers and components ofvaccines, can be produced according to the present methods to havecontrolled and particular characteristics. Use of the present gelatinsthus permits vaccine makers an opportunity to fine-tune and minimizevariability in their production processes, as well as to use recombinantgelatin in a more effective and more cost-efficient manner thancurrently available animal-source products are used.

[0246] It is specifically contemplated that the recombinant gelatins ofthe present invention can be used in any type of vaccine. For example,the present recombinant gelatins can be used in live attenuated,inactivated, and subunit vaccines, in single-dosage and combinationvaccines, in virus-based, bacterial-based, parasite-based, and nucleicacid vaccines, including synthetic and semi-synthetic vaccines.

[0247] The present vaccines can be formulated in any fashion appropriatefor the particular vaccine, for example, as liquid or as freeze-driedformulations. More particularly, the invention relates to compositionsthat stabilize liquid and lyophilized viral vaccines and protein-basedpharmaceuticals. Different types of vaccines, directed to variousmicroorganisms, including viruses, bacteria, and parasites, etc., arecontemplated. For example, the recombinant gelatins of the presentinvention can be used in the formulation of, e.g., live, inactivated,and subunit vaccines.

[0248] In one aspect, the present invention provides a subunit vaccinecomprising recombinant gelatin. In a further embodiment, the subunitvaccine comprises non- immunogenic recombinant gelatin. In oneembodiment, the recombinant gelatin can be recombinant human gelatin. Invarious embodiments, the recombinant gelatin is derived from hydrolysisof recombinant collagen, is produced directly from altered collagenconstructs, is a homogeneous mixture of uniform molecular weight, andhas the melting temperature most appropriate for the vaccine'sparticular formulation and delivery mechanism. In another embodiment,the recombinant human gelatin comprises a sequence selected from thegroup consisting of SEQ ID NOs:15 through 25, 30, 31, and 33.

[0249] The recombinant gelatins of the present invention can be used asstabilizers in any vaccine directed to any infective agent. Examples ofsuch vaccines include, but are not limited to, vaccines for vacinniavirus (smallpox), polio virus (Salk and Sabin), mumps, measles, rubella,diphtheria, tetanus, Varicella-Zoster (chicken pox/shingles), pertussis(whopping cough), Bacille Calmette-Guerin (BCG, tuberculosis),haemophilus influenzae meningitis, rabies, cholera, plague, Japaneseencephalitis virus, salmonella typhi, shigella, hepatitis A, hepatitisB, rotavirus, adenovirus, yellow fever, foot-and-mouth disease, herpessimplex virus, respiratory syncytial virus, rotavirus, Dengue, West Nilevirus, Turkey herpes virus (Marek's Disease), influenza, parainfluenza,respiratory syncytial virus (RSV), typhus, pneumonia, Lyme disease, andanthrax. The term vaccine as used herein includes reference to vaccinesto various infectious and autoimmune diseases and cancers that have beenor that will be developed, for example, vaccines to various infectiousand autoimmune diseases and cancers, e.g., vaccines to humanimmunodeficiency virus (HIV), hepatitis C virus (HCV), and malaria, andvaccines to breast, lung, colon, renal, bladder, and ovarian cancers.

[0250] The present invention encompasses formulations intended forvarious types of delivery. The vaccines can be formulated for injection,including, for example, subcutaneous, parenteral, e.g., intramuscular,and intravenous injection. In one embodiment, the vaccine is formulatedfor delivery to a mucosal surface, such as for oral or nasal delivery,in spray, liquid, or other forms. In a further aspect, such formulationsinclude mucosal absorption enhancers as appropriate. Vaccines formulatedas inhalants, e.g., for deep lung delivery, are specificallycontemplated. Such a vaccine could be formulated, for example, in finepowder form. The vaccines of the present invention could also beformulated for transdermal or liposomal delivery. Various formulationssuitable for, e.g., enteric release are specifically contemplated.

[0251] The vaccine formulations of the present invention can includevaccines intended as edible vaccines, such as food vaccines, in whichthe antigen is delivered in edible form, such as in transgenic plants,e.g., potatoes, is capable of inducing the appropriate protectiveresponse. (See, e.g., Tacket, C. O. et al. (2000) J. Infect. Dis.182:302-305; and Richter, L. J. et al. (2000) Nat. Biotech.18:1167-1171.) The vaccines of the present invention can be provided inthe form of transgenic plants, e.g., edible fruits, such as bananas orstrawberries.

[0252] Encapsulation of the present invention, for example, inmicrospheres, is specifically contemplated. (See, e.g., Moldoveanu, Z.et al. (1993) J. Inf Dis. 167:85-90.) Controlled release technologiesavailable to those in the art can be applied to obtain formulationsappropriate in, for example, various release forms, such as, e.g.,transdermal, pulmonary, and polymeric release forms are specificallycontemplated.

[0253] Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.Formulations for enteric release, etc., are also contemplated. Thecomposition may be administered in a local rather than a systemicmanner.

[0254] Techniques for various formulations and drug delivery systems areavailable in the art and are described in numerous sources. (See, e.g.,Remington's Pharmaceutical Sciences, supra.) The most effective andconvenient route of administration and the most appropriate formulationfor a particular situation can be readily determined by methods known inthe art.

[0255] The following examples explain the invention in more detail. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

EXAMPLES

[0256] Unless otherwise stated, the following materials and methods wereused in the examples of the present invention.

Example 1 Direct Expression of Recombinant Gelatins

[0257] Specific fragments of the α1(I) cDNA from human type I collagenwere amplified by PCR and cloned into the plasmid pPICZαA or pPIC9K(Invitrogen Corp., Carlsbad, Calif.). The specific PCR primers used incloning are set forth in Table 1 below. Specific recombinant gelatinsare identified in Table 2 as SEQ ID NOs: 15 through 25, and 30, 31, and33. These recombinant gelatins are additionally identified by referenceto human prepro-α1(I) collagen. (Genbank Accession No. CAA98968.) Theexpression plasmids used contained α1(I) cDNA sequences of differentsizes fused to the yeast mating factor alpha prepro secretion sequence.Other signal sequences known in the art can also be used, for example,the yeast invertase (SUC2), the yeast acid phosphatase (PHO) sequences,the native pro-collagen signal sequence, and the signal sequence forhuman serum albumin. A signal sequence that provides the optimal levelof expression for a specific gelatin fragment in a specific expressionsystem should be chosen. TABLE 1 SEQ ID NO: SEQUENCE 1GTATCTCTCGAGAAGAGAGAGGCTGAAGCTGGTCTGCC TGGTGCCAAGGGT 2TAGACTATTATCTCTCGCCAGCGGGACCAGCAGG 3GTATCTCTCGAGAAGAGAGAGGCTGAGGCTGGAGCTCA GGGACCCCCTGGC 4ATGCTCTAGATTATTACTTGTCACCAGGGGCACCA GCAGG 5GTATCTCTCGAGAAGAGAGAGGCTGAAGCTGGCCCCAT GGGTCCCTCTGGTCCT 6TGCTCTAGATCATTAAGCATCTCCCTTGGCACCATCCAA 7TGCTCTAGACTATTAAGGCGCGCCAGCATCACCCTTAG CACCATC 8TGCTCTAGATCATTAAGGCGCGCCAGGTTCACCGCTGT TACCCTTGGG 9TGCTCAGTCATTATCTCTCGCCTCTTGCTCCAGAGGG 10GTGCCCGTGGTCAGGCTGGTGTGATGGGATTCCCTGGA CCTAAAGGTGCTGCTTAAT 11CTAGATTAAGCAGCACCTTTAGGTCCAGGGAATCCCAT CACACCAGCCTGACCACGGGCACCAG 12ATGCTCTAGATTATTAAGGAGAACCGTCTCGTCCA GGGGA 13CTAGTCTAGATTATCTTGCTCCAGAGGGGCCAGGGGC 14CTAGTCTAGATTAGCGAGCACCTTTGGCTCCAGGAGC 32AGCTTCTAGATTATTAGGGAGGACCAGGGGGACCAGGA GGTCC

[0258] TABLE 2 SEQ ID AMINO MOLECULAR NO: PCR PRIMERS USED ACID SEQUENCEWEIGHT 15 SEQ ID NO:5 and SEQ ID NO.6 residue 179 to residue 280  9,447Da 16 SEQ ID NO 5 and SEQ ID NO 8 residue 179 to residue 439 23,276 Da17 SEQ ID NO:5 and SEQ ID NO 9 residue 179 to residue 679 44,737 Da 18SEQ ID NO 10 and SEQ ID NO 11 residue 531 to residue 589  5,250 Da 19 SEID NO: 1 and SEQ ID NO 2 residue 531 to residue 631  8,947 Da 20 SEQ IDNO 1 and SEQ ID NO 7 residue 531 to residue 715 16,483 Da 21 SEQ ID NO 1and SEQ ID NO 4 residue 531 to residue 781 22,373 Da 22 SEQ ID NO 1 andSEQ ID NO 12 residue 531 to residue 1030 44,216 Da 23 SEQ ID NO:3 andSEQ ID NO:7 residue 615 to residue 715  8,213 Da 24 SEQ ID NO 3 and SEQID NO 4 residue 615 to residue 781 14,943 Da 25 SEQ ID NO 3 and SEQ IDNO:12 residue 615 to residue 1030 36,785 Da 30 SEQ ID NO 3 and SEQ ID NO13 residue 615 to residue 676  5,517 Da 31 SEQ ID NO 3 and SEQ ID NO 14residue 615 to residue 865 22,126 Da 33 SEQ ID NO:1 and SEQ ID NO 32residue 531 to residue 1192 ˜65 kDa

[0259] The expression plasmids were introduced into Pichia pastoriscells by electroporation, and transformants were selected bycomplementation of a his4 auxotrophy (pPIC9K vectors) or by resistanceto zeocin (pPICZαA vectors). Recombinant protein expression wasregulated by the methanol-inducible alcohol oxidase promoter (P_(AOX1)).The Pichia pastoris host cells contained integrated copies of the α andβ subunits of human prolyl 4-hydroxylase (P4H), the enzyme responsiblefor the post-translational synthesis of hydroxyproline in collagen, andhave been previously described. (See, e.g., Vuorela, M. et al. (1997)EMBO J 16:6702-6712.)

[0260] The yeast strains were grown in buffered minimal glycerol media,and recombinant protein expression was induced using the same media withmethanol (0.5%) substituted for glycerol as the carbon source, asdescribed in the Invitrogen Pichia Expression Manual. Gelatin-producingstrains were identified by 10-20% Tricine SDS-PAGE analysis ofconditioned media and prolyl 4-hydroxylase activity in extracts fromshake flask cultures. Co-expression of prolyl 4-hydroxylase and thecollagen fragments resulted in the expression of recombinant gelatinswith native human sequences.

[0261] The fragments were expressed and secreted into the media.Recombinant gelatin was recovered and purified from the media by acetoneprecipitation, anion or cation exchange chromatography, or anycombination thereof. Acetone precipitation was performed at 4° C. byaddition of acetone to cell-free culture supernatants to a finalconcentration of 40%. The resulting precipitate, consisting primarily ofendogenous yeast proteins, was collected by centrifugation. Acetone wasthen added to this supernatant to a final concentration of 80%, causingthe gelatin to precipitate, which was then collected by centrifugation,dialyzed overnight against water, and lyophilized. High purity gelatinwas obtained by a combination of anion and cation exchangechromatography. Chromatographic purifications were performed at roomtemperature.

[0262] Estimation of the sizes of collagenous proteins byelectrophoresis, compared to calculation of molecular weight based onamino acid composition, is known in the art (Butkowski et al. (1982)Methods Enzymol 82:410-423) N-terminal sequence analysis of therecombinant gelatins demonstrated correct processing of the preprosequence which was fused to the gelatin fragments in order to direct theprotein to the yeast secretory pathway. The gelatins produced in thissystem contained only sequences derived from human collagen.Additionally, the recombinant gelatins represented the major componentof the conditioned media, as Pichia pastoris cells secrete very fewproteins.

[0263] The expressed recombinant gelatins were of discrete sizes,ranging from about 5 kDa to about 65 kDa as measured on SDS-PAGE,corresponding to gelatins of ˜5 kDa (lane 2, SEQ ID NO:18), ˜10 kDa(lane 3, SEQ ID NO:19), ˜16 kDa (lane 4, SEQ ID NO:24), ˜18 kDa (lane 5,SEQ ID NO:20), ˜20 kDa (lane 6, SEQ ID NO:28) (also having a calculatedmolecular weight of 17,914 Da, not set forth in Table 1), ˜33 kDa (lane7, SEQ ID NO:27) (also having a calculated molecular weight of 29,625Da, not set forth in Table 1), ˜41 kDa (lane 8, SEQ ID NO:25), and ˜50kDa (lane 9, SEQ ID NO:22), as indicated in FIG. 1 (lane 10 representshydrolyzed recombinant human collagen type I, prepared as described inExample 10).

Example 2 Human Recombinant Gelatins Support Cell Attachment Activity

[0264] The recombinant human gelatin fragments of the present inventiondemonstrated in vitro cell attachment activity. In the following assay,96-well Maxisorp plates (Nunc) were coated with the followingrecombinant human gelatin domains from the α1 chain of human type Icollagen, as described in Example 1 and listed in Table 2: SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. VITROGEN bovine collagen(Cohesion Technologies; Palo Alto, Calif.) and bovine serum albuminserved as positive and negative controls, respectively. Each of theproteins was diluted to 0.1 mg/ml in 0.1 M NaHCO₃, pH 10.0, and theplates coated overnight at 4° C. Human foreskin fibroblasts (HFF) orhuman umbilical vein endothelial cells (HUVEC, from Clonetics, passage5), were seeded onto the coated plates and incubated for 60 minutes at37° C. Experiments were performed in triplicate.

[0265] The degree of cell attachment was then measured using ReagentWST-1, the absorbance of which was read at 450 mM in an ELISA reader.FIG. 2A shows that recombinant human gelatins supported HFF attachmentto Maxisorp plates, and, for these cells, attachment was directlyproportional to the molecular weight of the recombinant human gelatincoated in each well. Specifically, the recombinant gelatins of SEQ IDNO: 19, SEQ ID NO:20, and SEQ ID NO:21 supported HFF attachment to ahigher extent than that seen with BSA. FIG. 2B shows that the differentrecombinant human gelatins supported endothelial cell attachment. Cellattachment activity was also demonstrated with recombinant human gelatinprepared by thermal hydrolysis of recombinant human collagen (describedbelow in Example 9), using recombinant gelatins having molecular weightranges of 0-30 kDa and 0-50 kDa.

Example 3 Identification of a Proteolytically Stable Gelatin Fragment

[0266] Recombinant gelatin fragments were found to be proteolyticallymodified during their expression and accumulation in the media ofrecombinant Pichia pastoris cells. Expression of several differentportions of the helical domain of the α1 chain of type I collagen leadto the identification of a recombinant gelatin that had superiorstability with respect to proteolysis. Three different gelatin fragmentswere cloned into plasmid pPICZαA, and their relative stabilitiesevaluated during recombinant protein expression in Pichia pastoriscells.

[0267] The first strain used is described above in Example 2,corresponding to SEQ ID NO:19. Additional strains were created usingplasmids encoding human α1 (I) helical domain amino acid residues179-280 (SEQ ID NO:15) and 615-715 (SEQ ID NO:23). These recombinantgelatins were constructed as described in Example 1, using primers SEQID NO:5 and SEQ ID NO:6, and SEQ ID NO:3 and SEQ ID NO:7. The PCRproducts were digested with XhoI and XbaI, cloned, and prepared forelectroporation as described above. The strains were grown, proteinexpression induced, and the expressed gelatin fragments compared bySDS-PAGE. FIG. 3 shows that the recombinant gelatin of SEQ ID NO:15(lane 2) and the recombinant gelatin of SEQ ID NO:19 (lane 3) underwentproteolysis, while the recombinant gelatin of SEQ ID NO:23 (lane 4)remained completely intact. This result demonstrated that recombinantgelatin fragments of the present invention could be produced which havesuperior stability.

Example 4 Expression of Hydroxylated Recombinant Human Gelatin

[0268] Prolyl 4-hydroxylase activity has not been detected in yeast. APichia pastoris strain has been engineered to express active prolyl4-hydroxylase and has been used previously to produce hydroxylatedcollagen. (See U.S. Pat. No. 5,593,859.) To express hydroxylatedrecombinant human gelatin, this strain was transformed with a gelatinexpression cassette encoding 100 amino acids of a recombinant of humanα1 (I) collagen (SEQ ID NO:19, Table 2), generated by PCR using theprimers SEQ ID NO:1 and SEQ ID NO:2. The PCR DNA product (˜330 bp) wasdigested with XhoI-XbaI and ligated into the XhoI-XbaI sites of pPICZαA(Invitrogen), creating plasmid pDO7.

[0269] A 1048 bp Cel II-AgeI fragment was isolated from pDO7 whichcontained the 3′ portion of the AOX1 promoter region, the mating factoralpha secretion signal, the recombinant gelatin of SEQ ID NO:19, thepolylinker sequence from pPICZαA, and 56 base pairs of the AOX1transcription terminator. This fragment was ligated into the Cel II-AgeIsites of pPIC9K (Invitrogen) to create pDO41. Pichia pastoris strain αβ8(his4) was transformed with StuI-linearized plasmid pDO41 byelectroporation, plated on minimal dextrose plates, and transformantswere selected that complemented the his4 auxotrophy. Approximately 20his⁺ transformants were grown and induced with methanol as described inExample 1. Strains that expressed SEQ ID NO:19 were identified bySDS-PAGE analysis of the conditioned media. (FIG. 4.)

[0270] Recombinant gelatin fragments from positive strains were purifiedfrom the media by acetone precipitation, and analyzed further by aminoacid analysis, as described, e.g., in Hare, PE. (1977) Methods inEnzymology 47:3-18. Amino acid analysis of the gelatin product from oneof the strains demonstrated the presence of hydroxyproline in thesecreted recombinant gelatins. The ratio of hydroxyproline to prolinewas determined to be 0.29 in gelatin isolated from the strain shown inshown in FIG. 4, isolate #2, indicating co-expression of gelatin andprolyl 4-hydroxylase.

[0271] Non-hydroxylated recombinant gelatins were expressed and purifiedfrom a Pichia pastoris strain that does not express prolyl4-hydroxylase. Proline residues within this recombinant gelatin weresubsequently converted to hydroxyproline residues in vitro using prolyl4-hydroxylase enzyme activity. A gelatin expression plasmid was createdby PCR using primers SEQ ID NO:3 and SEQ ID NO:4, leading to theexpression of recombinant gelatin of SEQ ID NO:24. The 525 base pair PCRproduct was purified and digested with XhoI-XbaI and ligated toXhoI-XbaI digested pPICZαA. The plasmid was linearized with PmeI andelectroporated into Pichia pastoris strain X-33 (Invitrogen).Transformants were selected by growth on YPD plates containing 500 μg/mlzeocin. Strains were tested for gelatin expression as described aboveand recombinant non-hydroxylated gelatin was purified from the media ofa positive isolate. Conditioned media was concentrated 10-fold bypressure dialysis using a 10 kDa molecular weight cut-off membrane, andthe sample was dialyzed against Buffer A (50 mM Tris-HCl pH 9.0, 50 mMNaCl). The dialyzed material was chromatographed on a Q-sepharose columnequilibrated in Buffer A. Gelatin does not bind to this column underthese conditions, and therefore, was present in the flow-throughfraction. The majority of the contaminating yeast proteins bound to thecolumn and eluted with Buffer B (Buffer A+0.5 M NaCl).

[0272] The flow-through fraction was dialyzed against 50 mM sodiumacetate, pH 4.5, and the recombinant gelatin further purified on aSP-sepharose column equilibrated in the same buffer. The recombinantgelatin bound to the column, and was step-eluted with 0.2 M NaCl. Thepurified gelatin, at 1 mg/ml, was heat denatured (100° C. for 10minutes) and mixed with purified P4H at a enzyme to substrate ratio of1:30 in the presence of the following components: 50 mM Tris-HCl pH 7.8,2 mM ascorbate, 2 mM α-ketoglutarate, 0.1 mM FeSO_(4,) 10 μM DTT, 10mg/ml bovine serum albumin, and 100 units of catalase (Sigma ChemicalCo., St Louis, Mo.). (See, e.g., Kivirikko, K. I. and Myllyla, R. (1982)Methods in Enzymology 82:245-304; and Vuori, K., et. al. (1992) Proc.Natl. Acad. Sci. 89:7467-7470.) The reaction was allowed to proceed at37° C. for 16 hours.

[0273] The recombinant gelatin was then purified by chromatography onQ-sepharose as described above. The bound proteins were eluted from thecolumn with 0.5 M NaCl and collected. (FIG. 5, lanes 7, 8, and 9.) Theflow-through and eluate fractions were analyzed by SDS-PAGE todemonstrate the purity of the recovered gelatin. (FIG. 5.) Amino acidanalysis of the gelatin was performed following dialysis of theflow-through fractions. (FIG. 5; lanes 3 through 6.) The amino acidanalysis showed that the gelatin was 87% hydroxylated. Hydroxylation of100% is achieved when the number of moles of hydroxyproline/moles ofproline+moles of hydroxyproline in gelatin equals 0.5.

Example 5 Stability of Gelatins in the Presence or Absence of Prolyl4-hydroxylase

[0274] An 18 kDa recombinant gelatin (SEQ ID NO:20) was expressedaccording to the methods described above. The expressed fragments wereanalyzed by gel electrophoresis. Recombinant gelatin expressed in thepresence of prolyl 4-hydroxylase had markedly greater stability than thegelatin expressed in the absence of prolyl 4-hydroxylase. (See FIG. 6.)

[0275] A role of proline hydroxylation on recombinant human gelatinstability and an enhancement of stability was found in prolyl4-hydroxylase-expressing Pichia pastoris strains. A plasmid encoding SEQID NO:20 (pDO32) was constructed by PCR using primers SEQ ID NO:1 andSEQ ID NO:7. The PCR product was purified, digested, and cloned asdescribed above. The same α1 (I) CDNA fragment was expressed in hostcells lacking prolyl hydroxylase, and in host cells containing the α andβ prolyl 4-hydroxylase subunits. Three Pichia pastoris strains wereelectroporated with PmeI-linearized pDO32: strain X-33 (wild-type Pichiapastoris), two prolyl 4-hydroxylase-expression strains: strain P4H-2,and strain αβ8, as described in the U.S. Pat. No. 5,593,859 and inVourela et al. (1997) EMBO J 16:6702-6712.

[0276] Transformants were selected by resistance to 500 μg/ml zeocin.Eight isolates from each transformation were grown and induced asdescribed, and the stability of the expressed recombinant human gelatinwas analyzed by SDS-PAGE. (See FIG. 6.) In wild-type Pichia pastorisstrain X-33, approximately equimolar amounts of intact recombinantgelatin and a proteolytic fragment (which migrated just below therecombinant gelatin on the gel, indicated by the arrow at the right ofthe figure) were observed. (FIG. 6, strain X-33.) In both strains thatco-express prolyl 4-hydroxylase, the amount of the proteolytic fragmentwas significantly reduced, demonstrating that co-expression of prolyl4-hydroxylase along with recombinant human gelatin enhanced gelatinstability by substantially reducing proteolysis of the gelatin. (FIG. 6,strain P4H-2 and strain αβ8.)

Example 6 Enhanced Recombinant Human Gelatin Expression bySupplementation of Expression Media

[0277] Previous reports have indicated that casamino acid-supplementedmedia decreased the amount of proteolysis seen during expression ofcertain proteins in Pichia pastoris. (Clare, J. J. et al. (1991) Gene105:202-215.) The breakdown of the present recombinant human gelatinexpressed in Pichia pastoris was measured following enrichment of theexpression media with various supplements. In this particular study, thePichia pastoris strain αβ8 described in Example 5, which expressedrecombinant human gelatin fragment SEQ ID NO:20 was employed. (Example 5and Table 2.) Recombinant gelatin was induced in media supplemented witha range of concentrations (0-2%) of various supplemental components,including casamino acids, casitone, yeast extract, peptone, peptamin,tryptone, Gelatone, lactalbumin, and soytone. Several formulations,including lactalbumin hydrolysate, soytone, casitone, and peptamin(Difco Laboratories, Detroit, Mich.) increased recombinant gelatinexpression levels. (FIG. 7, lactalbumin and soytone.)

[0278] These results indicate that specific media supplements employedduring the expression of recombinant gelatins can lead to increasedproduction. In one aspect, the use of soytone as a media supplementprovided a plant-derived (rather than animal-derived) media componentthat lead to increased expression of recombinant gelatin. This wouldprovide an animal material-free environment for production ofrecombinant gelatin that could be used in a variety of applications.

Example 7 Cross-linking of Recombinant Human Gelatins

[0279] A slurry of recombinant human collagen (obtained as described inU.S. Pat. No. 5,593,859) was prepared by dissolving 10.8 mg ofrecombinant human collagen type I in 5 ml of water, followed by dialysisagainst 20 mM sodium phosphate, pH 7.2. The final recombinant humancollagen concentration of the slurry was approximately 2 mg/ml.Preparation of cross-linked recombinant human gelatin was performed byadding 10 μl or 5 μl of a 20% solution of1-ethyl-3-(3-dimethlyaminopropyl) carbodiimide hydrochloride (EDC,Pierce Chemical Co.) to 1 ml of the recombinant human collagen slurrydescribed above. The cross-linking reaction occurred overnight at roomtemperature. Unreacted EDC was removed by dialysis against water.

[0280] The resulting cross-linked recombinant human gelatins wereanalyzed by 6% glycine SDS-PAGE analysis. FIG. 8 shows an SDS-PAGEcomparison of recombinant human gelatin (lane 6, labeled UNL-5-4),cross-linked recombinant human gelatin (lane 5, labeled UNL 5-4, 0.1%EDC; lane 4, labeled UNL 5-4, 0.2% EDC), commercially available hardcapsule gelatin (lane 3), and commercially available gelatin (Type A,from porcine skin, approximately 300 Bloom, lane 2) obtained from SigmaChemical Co. As shown in the SDS-PAGE analysis of FIG. 8, the commercialcapsule gelatin and Sigma gelatin contained α-chain (molecular weight ofapproximately 110 kDa) as a major component, as well as a smear ofhigher molecular weight gelatin components (with molecular weightranging from approximately 200-250 kDa). The recombinant human collagenwas composed of α-chain only. Following cross-linking, however, thecross-linked recombinant gelatin was composed of α-chain as well as asmear of higher molecular weight gelatins, similar to that observed incommercial gelatin and commercial capsule gelatin. This indicated thatrecombinant human gelatins displaying a molecular weight distributionsimilar to that of commercial capsule gelatins could be produced bycross-linking recombinant human collagen. Cross-linked recombinantgelatins would have use in applications in which increased gel strengthand increased viscosity would be desirable.

Example 8 Endotoxin Levels of Commercially Available Gelatin and SolubleRecombinant Human Gelatin

[0281] Endotoxin levels of soluble gelatin obtained commercially fromKind & Knox (K&K) and the recombinant human gelatins of the presentinvention (made as described in Example 9) were determined using theLimulus Ameobocyte Lysate test, as known in the art. (See, e.g.,Friberger, P. et al. (1987) Prog. Clin. Biol. Res. 231:149-169.) Threedifferent gelatin concentrations were examined. As shown in Table 3, therecombinant human gelatins generated by thermal hydrolysis ofrecombinant human collagen type I (rhcI) of the present invention werevirtually endotoxin-free. The endotoxin levels of commercially availablematerials were about 1 to 1.5 EU/mg of protein. The methods forproducing gelatin as described in the present invention resulted ingelatins having substantially lower endotoxin levels, by two to threeorders of magnitude, than those of the commercial preparations. Such lowendotoxin levels make the recombinant gelatins of the present inventionespecially attractive for use in certain applications, such as use inpharmaceutical stabilization. TABLE 3 Gelatin Concentration K&K GelatinRecombinant Human (mg/ml) (EU/mg) Gelatin (EU/mg) 3 1.03 <0.005 1.5 1.41<0.005 0.75 1.29 <0.006

Example 9 Derivation of Gelatins by Thermal and Acid Hydrolysis

[0282] Hydrolysis procedures (acid, thermal, and enzymatic) weredeveloped to produce soluble recombinant human gelatins with molecularweight distributions similar to those of currently available solubleanimal-derived gelatins, used, for example, as stabilizers in theformulation of vaccines. For these experiments, intact recombinant humancollagen type I and type III were used as starting materials. By varyingthe hydrolysis conditions, it was possible to vary the molecular weightsof the final materials, producing materials of defined molecularweights.

[0283] Molecular weight distribution of commercially available gelatins:

[0284] These recombinant human gelatins were compared againstcommercially available gelatins. Four low molecular weight gelatinsamples produced by Leiner Davis, Great Lake, Kind & Knox, and Dynagel,were obtained for characterization. All gelatins examined were solubleat room temperature. The molecular weight distributions of the gelatinson a Tricine SDS-PAGE gel are shown on FIG. 9 and listed in Table 4. Thegel profiles indicated the molecular weight distributions ofcommercially available gelatins were approximately 0-55 kDa, with theexception of the Dynagel-1 sample, which had a molecular weightdistribution of 0-30 kDa. The gel profiles also revealed two patterns ofmolecular weight distribution. In one example, derived from the samplesfrom Leiner Davis and Great Lakes, several discrete molecular bands wereobserved by SDS-PAGE. The pattern in the second example, derived fromthe Dynagel and Kind &Knox samples, showed a continuous distribution ofmaterial on the gel, with no discrete banding. The molecular weightdistributions of Dynagel-1 and Dynagel-2 were quite different, despitebeing produced by the same manufacturer for the same application. Thisresult indicated that batch-to-batch variation could be quitesignificant in currently available gelatins. TABLE 4 Maximum ApparentRelative Molecular Weight Molecular Weight* Company Mobility (Da)Distribution (Da) K & K 0.3410 70,000 0-55,500 Leiner Davis 0.341070,000 0-55,500 Great Lake 0.3693 60,000 0-47,600 Sol-U-Por, #1 0.348365,000 0-51,600 Sol-U-Por, #2 0.4972 37,000 0-29,400

[0285] Heat hydrolysis of gelatins was performed as follows. Thecommercially available dry gelatins were dissolved in 40°-50° C. waterto make a 5% gelatin solution. The pH of the solution was adjusted witheither 0.1N NaOH or 0.1N HCl in preparation for heat hydrolysis. Bothtype I and type III recombinant human collagens were expressed in Pichiapastoris and purified, as described in U.S. Pat. No. 5,593,859. Thefinal recombinant human collagen was dissolved in 10 mM HCl, dialyzedagainst water, and lyophilized. The lyophilized recombinant humancollagen was dissolved in 40°-50° C. water to make a 3% solution. The pHof the solution was adjusted as indicated below prior to heathydrolysis.

[0286] Heat hydrolysis was performed in 1 ml Reacti-Vials (Pierce). Thehydrolysis temperature varied from 100° C. to 150° C., depending on theexperiment. The pH of the hydrolysis solution varied from pH 2 to pH 7,as indicated. The hydrolysis time was also varied from one to thirty-twohours, depending on the temperature and pH of the solution. The gelatinhydrolysates were sampled at various time intervals and analyzed bySDS-PAGE.

[0287] Hydrolysis of Commercially Available Gelatins at 120 ° C.:

[0288] Samples of high molecular weight gelatin from Sigma (Type A fromporcine skin, 250 kDa) were dissolved in six different pH solutions (5%gelatin) and hydrolyzed at 120° C. The pH 2 and pH 3 solutions werehydrolyzed for two and a half hours and sampled every half hour. The pH4 solutions were hydrolyzed for five hours and sampled every hour. ThepH 5, pH 6, and pH 7 solutions were hydrolyzed for 24 hours and sampledevery two hours after 14 hours of hydrolysis.

[0289] The hydrolysis patterns were analyzed on Tricine 10-20% SDS-gelsas shown in FIG. 10A, 10B, 10C, 10D, 10E, and 10F. The gel profiles showthat the lower the pH of the solution, the more quickly the hydrolysisof the gelatin occurred. The gel profiles also revealed two hydrolysispatterns among the hydrolysates. One pattern showed several discretemolecular bands on the gel (see the acid hydrolysis results of the pH 2and pH 3 solutions FIG. 10A and 10B), while the other pattern showed acontinuous distribution of material in the gel (see the hydrolysisresults of the pH 4, pH 5, pH 6, and pH 7 solutions, FIG. 10C, 10D, 10E,and 10F).

[0290] These results showed that the process outlined above, orvariations thereof, produced two different types of material, as seen inthe analysis of the commercially available gelatins (discrete bands vs.a continuous distribution of material on SDS-PAGE). These experimentalresults also indicated that heat degradation of high molecular weightgelatin generated various sizes of soluble gelatins. Table 5 shows themolecular weight distributions obtained using Sigma Gelatin, followinghydrolysis at 120° C. in pH 6.0 solution. TABLE 5 Hydrolysis TimeRelative Max. App. Mol. Molecular Weight (hr) Mobility Weight (Da)Distribution (Da) 4 0.2356 140,000  0-111,000 8 0.2890 90,000 0-71,40011.5 0.3372 75,000 0-59,500 16 0.3837 47,000 0-37,300 20 0.4186 40,0000-31,700 24 0.4525 33,000 0-26,200

[0291] Hydrolysis of Commercially Available Gelatins at 150° C.:

[0292] Samples of high molecular weight gelatin from Sigma (Type A fromporcine skin 250 kDa) were dissolved in four different pH solutions (5%gelatin) and hydrolyzed at 150° C. up to ten hours. The hydrolysateswere sampled every two hours for analysis. The hydrolysis patterns wereanalyzed by Tricine 10-20% SDS-PAGE gels as shown in FIGS. 11A, 11B,11C, and 11D. The gel profiles indicated that the degradation of gelatinoccurred much more rapidly at 150° C. than at 120° C. Additionally,hydrolysis of gelatins performed at 150° C. produced gelatin fragmentsof lower molecular weights. Table 6 shows the molecular weightdistributions of Sigma Gelatin, following hydrolysis at 150° C. in pH6.0 solution. TABLE 6 Hydrolysis Time Relative Max. App. Mol. MolecularWeight (hr) Mobility Weight (Da) Distribution (Da) 2.5 0.2833 95,0000-75,400 4.5 0.4555 41,000 0-32,500 6 0.5277 32,000 0-25,400 8 0.583324,000 0-19,000 10 0.6611 15,000 0-11,900

Example 10 Acid and Thermal Hydrolysis of Recombinant Human Collagen Iand III

[0293] Recombinant human collagen type I was hydrolyzed at 120° C. forup to 8 hours under neutral pH conditions (pH 7), or up to 3 hours inacidic pH conditions (pH 2). Recombinant human collagen type III wasalso hydrolyzed at 120° C. for up to six hours in both neutral andacidic conditions. Hydrolysis was performed as described in Example 9.The human recombinant type I and type III hydrolysates were analyzed byTricine 10-20% SDS-PAGE gels, shown in FIGS. 12A and 12B. The SDS-PAGEgel patterns indicated that the heat hydrolysis of recombinant humancollagen was identical to the hydrolysis patterns of high molecularweight gelatins derived from natural sources. (FIG. 9, FIGS. 10A through10F, and FIGS. 11A through 11D, to FIGS. 12A and 12B.) Similar to thehydrolysis of natural gelatins (pH 7), the acid hydrolysates ofrecombinant human collagen showed several discrete molecular weightbands, while the neutral hydrolysates showed a more continuous molecularweight distribution. The molecular weight distribution of the neutralhydrolysates of recombinant human gelatin was around 0-70 kDa after sixto eight hours of heat degradation. The hydrolysis under acidicconditions occurred much faster. The molecular weight distributions ofthe acidic hydrolysates of recombinant human gelatin were much narrower,around 0-10 kDa, after two to three hours of heat treatment.

[0294] As a further refinement of the heat hydrolyzed recombinant humangelatins discussed, we have demonstrated the utility of a yeastmulti-gene recombinant expression methodology for the production ofhuman gelatins with discrete fragments of the α1 (I) chain of human typeI collagen. This technology allowed us to produce well-defined, highlyhomogeneous gelatin fragments ranging in size from 6-65 kDa. Thispresents unsurpassed flexibility in terms of the size and biophysicalproperties of the gelatin that can be used for specific applications.

Example 11 Enzymatic Hydrolysis of Recombinant Human Collagen Type I

[0295] Recombinant human collagen type I was hydrolyzed enzymatically,using the proteases set forth in Table 7. Recombinant human collagentype I was incubated with each enzyme at 37° C., using a substrate toenzyme ratio (w/w) as indicated in Table 7. The human recombinant type Ihydrolysates obtained by treatment were analyzed by Tricine 10-20%SDS-PAGE gels. The results obtained from papain and protease X treatmentare shown in FIG. 13. The SDS-PAGE gel patterns indicated that theenzymatic hydrolysis of recombinant human collagen lead to differentmolecular weight distributions of the gelatins. Enzymatic hydrolysisusing papain resulted in a continuous hydrolysis pattern, as indicatedin FIG. 13 and in Table 7, while hydrolysis using protease X resulted inseveral discrete molecular weight bands. As indicated in Table 7, therecombinant gelatins produced by this method had different hydrolysispatterns as a result of the particular enzymatic hydrolysis treatment.This presents great flexibility in producing sizes and biophysicalproperties of the gelatin that can be used for specific applications.TABLE 7 Enzyme Activity / Substrate to Hydrolysis Enzyme mg ProteinEnzyme Ratio Pattern Chymo-  1 U @ 37° C., pH 6.5 500:1 Continuouspapain Bromelain  8 U @ 37° C., pH 4.6 5,000:1 Banding & ContinuousProtease VIII 12 U @ 37° C., pH 8.5 7,000:1 Banding Papain 17 U @ 37°C., pH 6.5 10,000:1 Continuous Protease X 42 U @ 37° C., pH 8.5 20,000:1Banding

Example 12 Antibodies to recombinant human collagen type I directedagainst different recombinant gelatins

[0296] Human recombinant type I collagen produced in the yeast Pichiapastoris was tested for its potential allergic reaction as a contactsensitizer on guinea pig, known as Maximization Study. After theduration of the study, the sera were collected to investigate theimmunogenecity of recombinant human type I collagen in guinea pig. Onegram of rhC I was immersed in 10 ml of either 0.9% Sodium ChlorideInjection (SCI) or sesame oil, and incubated for 72 hours at 37° C. Theextract was then centrifuge at 3000 rpm for 15 minutes and thesupernatant collected for dosing.

[0297] Hartley pigs were exposed to the test article and controlsolution by an induction phase. This phase involved three pairs ofintradermal (ID) injections on clipped areas. The first pair of IDinjections (cranial) consisted of an emulsion of Freud's CompleteAdjuvant (FCA) in an equal volume of SCI. The second pair of IDinjections (middle) consisted of the test extract (recombinant humantype I collagen). The third pair (caudal) consisted of an emulsion ofthe test extract article and equal volume of FCA. Positive and negativecontrol animals were treated in a similar manner as the test animals,except that the test extract was not included in the second and thirdpair of injections.

[0298] On the sixth day after ID injections, the test sites wereevaluated for evidence of irritation. The test sites were thenpretreated with 10% SLS in petroleum and massaged into the skin using aglass rod, and then left uncovered for 24 hours. On the seventh day, atopical application was administered on the shaved areas of each testanimals with 4.25 cm diameter disk of Whatman #3 filter paper soakedwith 0.4 ml the test article extract. Thirteen days after the topicalinduction application, the animals were challenged. An area on the rightside of each animal was clipped. On the next day, Hill Top chamberscontaining 0.3 ml of test extract, vehicle control extract, or positivecontrol solutions were applied to clipped areas and remained on theanimals for 24 hours. The dosing sites were scored for erythema andedema 24, 48, and 72 hours after removal of the chambers.

[0299] After 72 hours, the blood was collected and allowed to clot, thencentrifuged at 2800 rpm for 15 minutes. The serum was removed from eachtube and serum samples were stored at −70° C. until use.

[0300] Sera from the immunized Guinea pigs were then analyzed for thepresence of antibodies directed against recombinant human collagen typeI (rhcI), recombinant human collagen type III (rhcIII), vitrogen (vitr),and various fragments of recombinant human gelatins of the presentinvention, including 6 kDa (SEQ ID NO:18), 10 kDa (SEQ ID NO:19), 18 kDa(SEQ ID NO:20), 33 kDa (SEQ ID NO:27), 50 kDa (SEQ ID NO:22), and 65 kDa(SEQ ID NO:33) fragments. (See Table 2 and Example 1.) Recombinantcollagen and recombinant gelatin were electrophoresed on 8% Tris-Glycineor 10-20% Tricine SDS-PAGE gels. Western blot analysis was performedusing anti-serum from each of the Guinea pigs used in the study. FIG. 14shows that recombinant human type I collagen-specific antibodies werepresent in the sera of Guinea pigs immunized with recombinant human typeI collagen. No antibody reactivity to any of the recombinant gelatinsanalyzed by Western blot analysis was observed in any of the sera ofexamined. FIG. 14 shows Western blot results using the antisera from oneGuinea pig in the study. The sera from at least 4 different Guinea pigswere analyzed, each of which showed identical results to that disclosedin FIG. 14.

[0301] It was desirable to elucidate possible epitopes of the type Icollagen responsible for the antigenic response observed followinginjection of rhcI into Guinea pigs. Recombinant human collagen type Iwas separated into its α1(I) and α2(I) components following denaturationand column chromatography. Cyanogen bromide (CNBr) cleavage of the α1(I)chain of recombinant type I collagen and the α2(I) chain of recombinanttype I collagen was performed as described in Bornstein and Piez (1966)Biochemistry 5:3460. The intact α chains and the resulting peptidefragments were separated by SDS-PAGE and analyzed by Western blotanalysis for reactivity to the Guinea pig sera described above. FIG. 15Ashows a coomassie-stained SDS-PAGE of intact and CNBr-cleaved α1(I) andα2(I) chains of recombinant human type I collagen. Western blot analysisshowed that the Guinea pig antisera reactive to rhcI were directedagainst the α2 chain of type I collagen and specific CNBr fragmentsthereof. No reactivity against the α1 chain of type I collagen wasdetected. (FIG. 15B.)

[0302] The Western blot analyses described above examined the reactivityof the Guinea pig sera to recombinant human type I collagen, CNBrfragments, and recombinant human gelatins by virtue of electrophoreticseparation on SDS-PAGE. To examine the reactivity of the Guinea pigantisera to these polypeptides under non-denatured conditions, a directELISA analysis was performed. (FIG. 16.) The data showed that the Guineapig antisera recognized the native conformation of rhcI. None of therecombinant gelatins of the present invention reacted with the Guineapig antisera by ELISA, regardless of whether the gelatin fragments werepresented before or after thermal denaturation. The rhcI was even morereactive in the ELISA if heat-denatured prior to analysis (data notshown). This indicated the polyclonal antibodies in the sera recognizedprimarily sequenced epitopes, rather than helical structures. Together,these results indicated that the concerns associated with having anantigenic site(s) present on human collagen type I, specifically to theα2 chain as shown in the current example, could be avoided by themethods of the present invention. The present invention thus providesmethods for generating recombinant gelatins lacking antigenic sites,which would be useful for specific applications in which gelatin of lowantigenicity is desired.

[0303] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the spirit and scope of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in the present art and related fieldsare intended to be within the scope of the following claims. Allreferences cited herein are incorporated by reference herein in theirentirety.

1 33 1 51 DNA human 1 gtatctctcg agaagagaga ggctgaagct ggtctgcctggtgccaaggg t 51 2 34 DNA human 2 tagactatta tctctcgcca gcgggaccag cagg34 3 51 DNA human 3 gtatctctcg agaagagaga ggctgaggct ggagctcagggaccccctgg c 51 4 40 DNA human 4 atgctctaga ttattacttg tcaccaggggcaccagcagg 40 5 54 DNA human 5 gtatctctcg agaagagaga ggctgaagctggccccatgg gtccctctgg tcct 54 6 39 DNA human 6 tgctctagat cattaagcatctcccttggc accatccaa 39 7 45 DNA human 7 tgctctagac tattaaggcgcgccagcatc acccttagca ccatc 45 8 48 DNA human 8 tgctctagat cattaaggcgcgccaggttc accgctgtta cccttggg 48 9 39 DNA human 9 tgctctagat cattatctctcgcctcttgc tccagaggg 39 10 57 DNA human 10 gtgcccgtgg tcaggctggtgtgatgggat tccctggacc taaaggtgct gcttaat 57 11 64 DNA human 11ctagattaag cagcaccttt aggtccaggg aatcccatca caccagcctg accacgggca 60ccag 64 12 40 DNA human 12 atgctctaga ttattaagga gaaccgtctc gtccagggga40 13 37 DNA human 13 ctagtctaga ttatcttgct ccagaggggc caggggc 37 14 37DNA human 14 ctagtctaga ttagcgagca cctttggctc caggagc 37 15 102 PRThuman 15 Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly1 5 10 15 Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro GlyGlu 20 25 30 Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly ProPro 35 40 45 Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg ProGly 50 55 60 Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro GlyThr 65 70 75 80 Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser GlyLeu Asp 85 90 95 Gly Ala Lys Gly Asp Ala 100 16 261 PRT human 16 Gly ProMet Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly 1 5 10 15 AlaPro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu 20 25 30 ProGly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro 35 40 45 GlyLys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly 50 55 60 GluArg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr 65 70 75 80Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu Asp 85 90 95Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly 100 105110 Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu 115120 125 Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg130 135 140 Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro ThrGly 145 150 155 160 Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly AlaLys Gly Glu 165 170 175 Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly ProGln Gly Val Arg 180 185 190 Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly AlaAla Gly Pro Ala Gly 195 200 205 Asn Pro Gly Ala Asp Gly Gln Pro Gly AlaLys Gly Ala Asn Gly Ala 210 215 220 Pro Gly Ile Ala Gly Ala Pro Gly PhePro Gly Ala Arg Gly Pro Ser 225 230 235 240 Gly Pro Gln Gly Pro Gly GlyPro Pro Gly Pro Lys Gly Asn Ser Gly 245 250 255 Glu Pro Gly Ala Pro 26017 501 PRT human 17 Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro GlyPro Pro Gly 1 5 10 15 Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro GlyGlu Pro Gly Glu 20 25 30 Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly ProPro Gly Pro Pro 35 40 45 Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys ProGly Arg Pro Gly 50 55 60 Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg GlyLeu Pro Gly Thr 65 70 75 80 Ala Gly Leu Pro Gly Met Lys Gly His Arg GlyPhe Ser Gly Leu Asp 85 90 95 Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly ProLys Gly Glu Pro Gly 100 105 110 Ser Pro Gly Glu Asn Gly Ala Pro Gly GlnMet Gly Pro Arg Gly Leu 115 120 125 Pro Gly Glu Arg Gly Arg Pro Gly AlaPro Gly Pro Ala Gly Ala Arg 130 135 140 Gly Asn Asp Gly Ala Thr Gly AlaAla Gly Pro Pro Gly Pro Thr Gly 145 150 155 160 Pro Ala Gly Pro Pro GlyPhe Pro Gly Ala Val Gly Ala Lys Gly Glu 165 170 175 Ala Gly Pro Gln GlyPro Arg Gly Ser Glu Gly Pro Gln Gly Val Arg 180 185 190 Gly Glu Pro GlyPro Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly 195 200 205 Asn Pro GlyAla Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala 210 215 220 Pro GlyIle Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser 225 230 235 240Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly 245 250255 Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu 260265 270 Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu275 280 285 Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu ProGly 290 295 300 Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe ProGly Ala 305 310 315 320 Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly GluArg Gly Ser Pro 325 330 335 Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly GluAla Gly Arg Pro Gly 340 345 350 Glu Ala Gly Leu Pro Gly Ala Lys Gly LeuThr Gly Ser Pro Gly Ser 355 360 365 Pro Gly Pro Asp Gly Lys Thr Gly ProPro Gly Pro Ala Gly Gln Asp 370 375 380 Gly Arg Pro Gly Pro Pro Gly ProPro Gly Ala Arg Gly Gln Ala Gly 385 390 395 400 Val Met Gly Phe Pro GlyPro Lys Gly Ala Ala Gly Glu Pro Gly Lys 405 410 415 Ala Gly Glu Arg GlyVal Pro Gly Pro Pro Gly Ala Val Gly Pro Ala 420 425 430 Gly Lys Asp GlyGlu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly 435 440 445 Pro Ala GlyGlu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe 450 455 460 Gln GlyLeu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro 465 470 475 480Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly 485 490495 Ala Arg Gly Glu Arg 500 18 59 PRT human 18 Glu Ala Gly Leu Pro GlyAla Lys Gly Leu Thr Gly Ser Pro Gly Ser 1 5 10 15 Pro Gly Pro Asp GlyLys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp 20 25 30 Gly Arg Pro Gly ProPro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly 35 40 45 Val Met Gly Phe ProGly Pro Lys Gly Ala Ala 50 55 19 101 PRT human 19 Glu Ala Gly Leu ProGly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser 1 5 10 15 Pro Gly Pro AspGly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp 20 25 30 Gly Arg Pro GlyPro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly 35 40 45 Val Met Gly PhePro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys 50 55 60 Ala Gly Glu ArgGly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala 65 70 75 80 Gly Lys AspGly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly 85 90 95 Pro Ala GlyGlu Arg 100 20 185 PRT human 20 Glu Ala Gly Leu Pro Gly Ala Lys Gly LeuThr Gly Ser Pro Gly Ser 1 5 10 15 Pro Gly Pro Asp Gly Lys Thr Gly ProPro Gly Pro Ala Gly Gln Asp 20 25 30 Gly Arg Pro Gly Pro Pro Gly Pro ProGly Ala Arg Gly Gln Ala Gly 35 40 45 Val Met Gly Phe Pro Gly Pro Lys GlyAla Ala Gly Glu Pro Gly Lys 50 55 60 Ala Gly Glu Arg Gly Val Pro Gly ProPro Gly Ala Val Gly Pro Ala 65 70 75 80 Gly Lys Asp Gly Glu Ala Gly AlaGln Gly Pro Pro Gly Pro Ala Gly 85 90 95 Pro Ala Gly Glu Arg Gly Glu GlnGly Pro Ala Gly Ser Pro Gly Phe 100 105 110 Gln Gly Leu Pro Gly Pro AlaGly Pro Pro Gly Glu Ala Gly Lys Pro 115 120 125 Gly Glu Gln Gly Val ProGly Asp Leu Gly Ala Pro Gly Pro Ser Gly 130 135 140 Ala Arg Gly Glu ArgGly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro 145 150 155 160 Pro Gly ProAla Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp 165 170 175 Gly AlaLys Gly Asp Ala Gly Ala Pro 180 185 21 251 PRT human 21 Glu Ala Gly LeuPro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser 1 5 10 15 Pro Gly ProAsp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp 20 25 30 Gly Arg ProGly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly 35 40 45 Val Met GlyPhe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys 50 55 60 Ala Gly GluArg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala 65 70 75 80 Gly LysAsp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly 85 90 95 Pro AlaGly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe 100 105 110 GlnGly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro 115 120 125Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly 130 135140 Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro 145150 155 160 Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly AsnAsp 165 170 175 Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly SerGln Gly 180 185 190 Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly AlaAla Gly Leu 195 200 205 Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly ProLys Gly Ala Asp 210 215 220 Gly Ser Pro Gly Lys Asp Gly Val Arg Gly LeuThr Gly Pro Ile Gly 225 230 235 240 Pro Pro Gly Pro Ala Gly Ala Pro GlyAsp Lys 245 250 22 500 PRT human 22 Glu Ala Gly Leu Pro Gly Ala Lys GlyLeu Thr Gly Ser Pro Gly Ser 1 5 10 15 Pro Gly Pro Asp Gly Lys Thr GlyPro Pro Gly Pro Ala Gly Gln Asp 20 25 30 Gly Arg Pro Gly Pro Pro Gly ProPro Gly Ala Arg Gly Gln Ala Gly 35 40 45 Val Met Gly Phe Pro Gly Pro LysGly Ala Ala Gly Glu Pro Gly Lys 50 55 60 Ala Gly Glu Arg Gly Val Pro GlyPro Pro Gly Ala Val Gly Pro Ala 65 70 75 80 Gly Lys Asp Gly Glu Ala GlyAla Gln Gly Pro Pro Gly Pro Ala Gly 85 90 95 Pro Ala Gly Glu Arg Gly GluGln Gly Pro Ala Gly Ser Pro Gly Phe 100 105 110 Gln Gly Leu Pro Gly ProAla Gly Pro Pro Gly Glu Ala Gly Lys Pro 115 120 125 Gly Glu Gln Gly ValPro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly 130 135 140 Ala Arg Gly GluArg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro 145 150 155 160 Pro GlyPro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp 165 170 175 GlyAla Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly 180 185 190Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu 195 200205 Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp 210215 220 Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly225 230 235 240 Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu SerGly Pro 245 250 255 Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala ProGly Asp Arg 260 265 270 Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe AlaGly Pro Pro Gly 275 280 285 Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu ProGly Asp Ala Gly Ala 290 295 300 Lys Gly Asp Ala Gly Pro Pro Gly Pro AlaGly Pro Ala Gly Pro Pro 305 310 315 320 Gly Pro Ile Gly Asn Val Gly AlaPro Gly Ala Lys Gly Ala Arg Gly 325 330 335 Ser Ala Gly Pro Pro Gly AlaThr Gly Phe Pro Gly Ala Ala Gly Arg 340 345 350 Val Gly Pro Pro Gly ProSer Gly Asn Ala Gly Pro Pro Gly Pro Pro 355 360 365 Gly Pro Ala Gly LysGlu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly 370 375 380 Pro Ala Gly ArgPro Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro 385 390 395 400 Ala GlyGlu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro 405 410 415 GlyThr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly 420 425 430Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro 435 440445 Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg 450455 460 Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly465 470 475 480 Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser ProGly Arg 485 490 495 Asp Gly Ser Pro 500 23 91 PRT human 23 Glu Ala GlyAla Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu 1 5 10 15 Arg GlyGlu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro 20 25 30 Gly ProAla Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly 35 40 45 Val ProGly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu 50 55 60 Arg GlyPhe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala 65 70 75 80 GlyPro Arg Gly Ala Asn Gly Ala Pro Gly Asn 85 90 24 167 PRT human 24 GluAla Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu 1 5 10 15Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro 20 25 30Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly 35 40 45Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu 50 55 60Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala 65 70 7580 Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly 85 9095 Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu 100105 110 Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys115 120 125 Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser ProGly 130 135 140 Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro ProGly Pro 145 150 155 160 Ala Gly Ala Pro Gly Asp Lys 165 25 416 PRT human25 Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu 1 510 15 Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro 2025 30 Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly 3540 45 Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu 5055 60 Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala 6570 75 80 Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly85 90 95 Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu100 105 110 Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly ProLys 115 120 125 Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly SerPro Gly 130 135 140 Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly ProPro Gly Pro 145 150 155 160 Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser GlyPro Ser Gly Pro Ala 165 170 175 Gly Pro Thr Gly Ala Arg Gly Ala Pro GlyAsp Arg Gly Glu Pro Gly 180 185 190 Pro Pro Gly Pro Ala Gly Phe Ala GlyPro Pro Gly Ala Asp Gly Gln 195 200 205 Pro Gly Ala Lys Gly Glu Pro GlyAsp Ala Gly Ala Lys Gly Asp Ala 210 215 220 Gly Pro Pro Gly Pro Ala GlyPro Ala Gly Pro Pro Gly Pro Ile Gly 225 230 235 240 Asn Val Gly Ala ProGly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro 245 250 255 Pro Gly Ala ThrGly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro 260 265 270 Gly Pro SerGly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly 275 280 285 Lys GluGly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg 290 295 300 ProGly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys 305 310 315320 Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly 325330 335 Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln340 345 350 Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly GluPro 355 360 365 Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly ProPro Gly 370 375 380 Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly GluSer Gly Arg 385 390 395 400 Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro GlyArg Asp Gly Ser Pro 405 410 415 26 510 PRT human 26 Gly Glu Arg Gly ValGln Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly 1 5 10 15 Ala Asn Gly AlaPro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala 20 25 30 Pro Gly Ala ProGly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro 35 40 45 Gly Glu Arg GlyAla Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly 50 55 60 Asp Ala Gly ProLys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val 65 70 75 80 Arg Gly LeuThr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro 85 90 95 Gly Asp LysGly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly 100 105 110 Ala ArgGly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro 115 120 125 AlaGly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys 130 135 140Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly 145 150155 160 Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly Ala165 170 175 Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly AlaThr 180 185 190 Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly ProSer Gly 195 200 205 Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly LysGlu Gly Gly 210 215 220 Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly ArgPro Gly Glu Val 225 230 235 240 Gly Pro Pro Gly Pro Pro Gly Pro Ala GlyGlu Lys Gly Ser Pro Gly 245 250 255 Ala Asp Gly Pro Ala Gly Ala Pro GlyThr Pro Gly Pro Gln Gly Ile 260 265 270 Ala Gly Gln Arg Gly Val Val GlyLeu Pro Gly Gln Arg Gly Glu Arg 275 280 285 Gly Phe Pro Gly Leu Pro GlyPro Ser Gly Glu Pro Gly Lys Gln Gly 290 295 300 Pro Ser Gly Ala Ser GlyGlu Arg Gly Pro Pro Gly Pro Met Gly Pro 305 310 315 320 Pro Gly Leu AlaGly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro 325 330 335 Ala Ala GluGly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly 340 345 350 Asp ArgGly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala 355 360 365 ProGly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg 370 375 380Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val Gly 385 390395 400 Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu405 410 415 Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly PheSer 420 425 430 Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly GluGln Gly 435 440 445 Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly ProPro Gly Ser 450 455 460 Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly LeuPro Gly Pro Ile 465 470 475 480 Gly Pro Pro Gly Pro Arg Gly Arg Thr GlyAsp Ala Gly Pro Val Gly 485 490 495 Pro Pro Gly Pro Pro Gly Pro Pro GlyPro Pro Gly Pro Pro 500 505 510 27 333 PRT human 27 Gly Ala Lys Gly AlaArg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly 1 5 10 15 Phe Pro Gly AlaAla Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn 20 25 30 Ala Gly Pro ProGly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys 35 40 45 Gly Pro Arg GlyGlu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly 50 55 60 Pro Pro Gly ProPro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala 65 70 75 80 Asp Gly ProAla Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala 85 90 95 Gly Gln ArgGly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly 100 105 110 Phe ProGly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro 115 120 125 SerGly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro 130 135 140Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala 145 150155 160 Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp165 170 175 Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly AlaPro 180 185 190 Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly AspArg Gly 195 200 205 Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly ProVal Gly Ala 210 215 220 Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly AspLys Gly Glu Thr 225 230 235 240 Gly Glu Gln Gly Asp Arg Gly Ile Lys GlyHis Arg Gly Phe Ser Gly 245 250 255 Leu Gln Gly Pro Pro Gly Pro Pro GlySer Pro Gly Glu Gln Gly Pro 260 265 270 Ser Gly Ala Ser Gly Pro Ala GlyPro Arg Gly Pro Pro Gly Ser Ala 275 280 285 Gly Ala Pro Gly Lys Asp GlyLeu Asn Gly Leu Pro Gly Pro Ile Gly 290 295 300 Pro Pro Gly Pro Arg GlyArg Thr Gly Asp Ala Gly Pro Val Gly Pro 305 310 315 320 Pro Gly Pro ProGly Pro Pro Gly Pro Pro Gly Pro Pro 325 330 28 200 PRT human 28 Glu ArgGly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro 1 5 10 15 ProGly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro 20 25 30 GlyArg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly 35 40 45 ProAla Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro 50 55 60 ValGly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala 65 70 75 80Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly 85 90 95Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp 100 105110 Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro 115120 125 Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly130 135 140 Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro GlyLys 145 150 155 160 Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro ProGly Pro Arg 165 170 175 Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro ProGly Pro Pro Gly 180 185 190 Pro Pro Gly Pro Pro Gly Pro Pro 195 200 29100 PRT human 29 Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg GlyIle Lys 1 5 10 15 Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro GlyPro Pro Gly 20 25 30 Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly ProAla Gly Pro 35 40 45 Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys AspGly Leu Asn 50 55 60 Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg GlyArg Thr Gly 65 70 75 80 Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro GlyPro Pro Gly Pro 85 90 95 Pro Gly Pro Pro 100 30 62 PRT human 30 Glu AlaGly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu 1 5 10 15 ArgGly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro 20 25 30 GlyPro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly 35 40 45 ValPro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg 50 55 60 31 251 PRThuman 31 Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu1 5 10 15 Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly LeuPro 20 25 30 Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu GlnGly 35 40 45 Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg GlyGlu 50 55 60 Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly ProAla 65 70 75 80 Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly AlaLys Gly 85 90 95 Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala ProGly Leu 100 105 110 Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu ProGly Pro Lys 115 120 125 Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala AspGly Ser Pro Gly 130 135 140 Lys Asp Gly Val Arg Gly Leu Thr Gly Pro IleGly Pro Pro Gly Pro 145 150 155 160 Ala Gly Ala Pro Gly Asp Lys Gly GluSer Gly Pro Ser Gly Pro Ala 165 170 175 Gly Pro Thr Gly Ala Arg Gly AlaPro Gly Asp Arg Gly Glu Pro Gly 180 185 190 Pro Pro Gly Pro Ala Gly PheAla Gly Pro Pro Gly Ala Asp Gly Gln 195 200 205 Pro Gly Ala Lys Gly GluPro Gly Asp Ala Gly Ala Lys Gly Asp Ala 210 215 220 Gly Pro Pro Gly ProAla Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly 225 230 235 240 Asn Val GlyAla Pro Gly Ala Lys Gly Ala Arg 245 250 32 43 DNA human 32 agcttctagattattaggga ggaccagggg gaccaggagg tcc 43 33 662 PRT human 33 Glu Ala GlyLeu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser 1 5 10 15 Pro GlyPro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp 20 25 30 Gly ArgPro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly 35 40 45 Val MetGly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys 50 55 60 Ala GlyGlu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala 65 70 75 80 GlyLys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly 85 90 95 ProAla Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe 100 105 110Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro 115 120125 Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly 130135 140 Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro145 150 155 160 Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro GlyAsn Asp 165 170 175 Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro GlySer Gln Gly 180 185 190 Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg GlyAla Ala Gly Leu 195 200 205 Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala GlyPro Lys Gly Ala Asp 210 215 220 Gly Ser Pro Gly Lys Asp Gly Val Arg GlyLeu Thr Gly Pro Ile Gly 225 230 235 240 Pro Pro Gly Pro Ala Gly Ala ProGly Asp Lys Gly Glu Ser Gly Pro 245 250 255 Ser Gly Pro Ala Gly Pro ThrGly Ala Arg Gly Ala Pro Gly Asp Arg 260 265 270 Gly Glu Pro Gly Pro ProGly Pro Ala Gly Phe Ala Gly Pro Pro Gly 275 280 285 Ala Asp Gly Gln ProGly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala 290 295 300 Lys Gly Asp AlaGly Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro 305 310 315 320 Gly ProIle Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly 325 330 335 SerAla Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg 340 345 350Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro 355 360365 Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly 370375 380 Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro385 390 395 400 Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala GlyAla Pro 405 410 415 Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg GlyVal Val Gly 420 425 430 Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro GlyLeu Pro Gly Pro 435 440 445 Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser GlyAla Ser Gly Glu Arg 450 455 460 Gly Pro Pro Gly Pro Met Gly Pro Pro GlyLeu Ala Gly Pro Pro Gly 465 470 475 480 Glu Ser Gly Arg Glu Gly Ala ProAla Ala Glu Gly Ser Pro Gly Arg 485 490 495 Asp Gly Ser Pro Gly Ala LysGly Asp Arg Gly Glu Thr Gly Pro Ala 500 505 510 Gly Pro Pro Gly Ala ProGly Ala Pro Gly Ala Pro Gly Pro Val Gly 515 520 525 Pro Ala Gly Lys SerGly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro 530 535 540 Ala Gly Pro ValGly Pro Val Gly Ala Arg Gly Pro Ala Gly Pro Gln 545 550 555 560 Gly ProArg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg Gly 565 570 575 IleLys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly Pro 580 585 590Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly Pro Ala 595 600605 Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly 610615 620 Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg625 630 635 640 Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro GlyPro Pro 645 650 655 Gly Pro Pro Gly Pro Pro 660

What is claimed is:
 1. A vaccine composition comprising: (a) arecombinant gelatin; and (b) an antigenic agent.
 2. The vaccinecomposition of claim 1, wherein the recombinant gelatin is recombinanthuman gelatin.
 3. The vaccine composition of claim 1, wherein therecombinant gelatin comprises a homogeneous mixture of recombinantgelatin polypeptides.
 4. The vaccine composition of claim 1, wherein therecombinant gelatin comprises a heterogeneous mixture of recombinantgelatin polypeptides.
 5. The vaccine composition of claim 1, wherein therecombinant gelatin is non- hydroxylated.
 6. The vaccine composition ofclaim 1, wherein the recombinant gelatin is hydroyxlated.
 7. The vaccinecomposition of claim 1, wherein the recombinant gelatin has a percentagehydroxylation selected from the group consisting of 20 to 80%, 30 to80%, 40 to 80%, 60 to 80%, 80 to 100%, 20 to 60%, 30 to 60%, 40 to 60%,20 to 30%, 20 to 40%, and 30 to 40%.
 8. The vaccine composition of claim1, wherein the recombinant gelatin is hydrolyzed.
 9. The vaccinecomposition of claim 1, wherein the recombinant gelatin is derived fromnon-native collagen sequence.
 10. The vaccine composition of claim 1,wherein the recombinant gelatin is obtained from one type of collagenfree of any other type of collagen.
 11. The vaccine composition of claim1, wherein the recombinant gelatin is proteolytically stable.
 12. Thevaccine composition of claim 1, wherein the recombinant gelatin isproduced by processing of recombinant collagen.
 13. The vaccinecomposition of claim 1, wherein the recombinant gelatin is produceddirectly from an altered collagen construct.
 14. The vaccine compositionof claim 1, wherein the recombinant gelatin has a molecular weight rangeselected from the group consisting of about 0 to 50 kDa, about 10 to 30kDa, about 30 to 50 kDa, about 10 to 70 kDa, about 50 kDa to 70 kDaabout 50 to 100 kDa, about 100 to 150 kDa, about 150 to 200 kDa, about200 to 250 kDa, about 250 to 300 kDa, and about 300 to 350 kDa.
 15. Thevaccine composition of claim 1, wherein the recombinant gelatin has amolecular weight selected from the group consisting of about 1 kDa,about 5 kDa, about 8 kDa, about 9 kDa, about 14 kDa, about 16 kDa, about22 kDa, about 23 kDa, about 44 kDa, and about 65 kDa.
 16. The vaccinecomposition of claim 1, wherein the recombinant gelatin comprises asequence selected from the group consisting of SEQ ID NOs: 15 through25, and 30, 31, and
 33. 17. The vaccine composition of claim 1, whereinthe recombinant gelatin is non-immunogenic.
 18. The vaccine compositionof claim 1, wherein the recombinant gelatin confers stability at ambienttemperatures.
 19. The vaccine composition of claim 1, wherein thevaccine composition is suitable for injectable delivery.
 20. The vaccinecomposition of claim 1, wherein the vaccine composition is suitable fornasal delivery.
 21. The vaccine composition of claim 1, wherein thevaccine composition is suitable for oral delivery.
 22. The vaccinecomposition of claim 1, wherein the vaccine composition is suitable fortransdermal delivery.
 23. The vaccine composition of claim 1, whereinthe vaccine composition is suitable for mucosal delivery.
 24. Thevaccine composition of claim 1, wherein the vaccine composition issuitable for deep lung delivery.
 25. The vaccine composition of claim 1,wherein the vaccine composition is liquid.
 26. The vaccine compositionof claim 1, wherein the vaccine composition is dry.
 27. The vaccinecomposition of claim 1, wherein the vaccine composition is lyophilized.28. The vaccine composition of claim 1, wherein the vaccine compositionis powdered.
 29. The vaccine composition of claim 1, wherein the vaccinecomposition is a spray.
 30. The vaccine composition of claim 1, whereinthe vaccine composition is an inhalant.
 31. The vaccine composition ofclaim 1, wherein the vaccine composition comprises a live vaccine. 32.The vaccine composition of claim 1, wherein the vaccine compositioncomprises an attenuated vaccine.
 33. The vaccine composition of claim 1,wherein the vaccine composition comprises an inactivated vaccine. 34.The vaccine composition of claim 1, wherein the vaccine compositioncomprises a subunit vaccine.
 35. The vaccine composition of claim 1,wherein the vaccine composition comprises a single dosage.
 36. Thevaccine composition of claim 1, wherein the vaccine compositioncomprises a multiple dosage.
 37. The vaccine composition of claim 1,wherein the vaccine composition comprises a conjugate vaccine.
 38. Thevaccine composition of claim 1, wherein the vaccine compositioncomprises a nucleic acid vaccine.
 39. The vaccine composition of claim38, wherein the nucleic acid vaccine is a DNA vaccine.
 40. The vaccinecomposition of claim 1, wherein the vaccine composition is a combinedvaccine.
 41. The vaccine composition of claim 1, wherein the vaccinecomposition comprises an acellular vaccine.
 42. The vaccine compositionof claim 1, wherein the vaccine composition comprises a vaccineformulated for the prevention of a disease selected from the groupconsisting of vacinnia virus (small pox), polio virus (Salk and Sabin),mumps, measles, rubella, diphtheria, tetanus, Varicella-Zoster (chickenpox/shingles), pertussis (whopping cough), Bacille Calmette-Guerin (BCG,tuberculosis), haemophilus influenzae meningitis, rabies, cholera,Japanese encephalitis virus, salmonella typhi, shigella, hepatitis A,hepatitis B, adenovirus, yellow fever, foot-and-mouth disease, herpessimplex virus, respiratory syncytial virus, rotavirus, Dengue, West Nilevirus, Turkey herpes virus (Marek's Disease), influenza, and anthrax.43. The vaccine composition of claim 1, wherein the recombinant gelatinhas an endotoxin level of below 1.000 EU/mg.
 44. The vaccine compositionof claim 1, wherein the recombinant gelatin has an endotoxin level ofbelow 0.500 EU/mg.
 45. The vaccine composition of claim 1, wherein therecombinant gelatin has an endotoxin level of below 0.050 EU/mg.
 46. Thevaccine composition of claim 1, wherein the recombinant gelatin has anendotoxin level of below 0.005 EU/mg.
 47. A method of producing avaccine composition comprising recombinant gelatin, the methodcomprising: (a) providing an antigenic agent; (b) providing arecombinant gelatin; and (c) combining the antigenic agent and therecombinant gelatin.
 48. A vaccine stabilizer comprising a recombinantgelatin.
 49. A vaccine stabilizer comprising a recombinant humangelatin.
 50. A method of inducing an immune response in a subject, themethod comprising administering the vaccine composition of claim 1 tothe subject.
 51. A method of stabilizing a vaccine composition, themethod comprising adding the vaccine stabilizer of claim 48 to a vaccinecomposition.
 52. A vaccination kit, the kit comprising: (a) a vaccinecomprising recombinant gelatin; and (b) a delivery device for deliveryof the vaccine.